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.
Intake of green tea may reduce the risk of breast cancer; polyphenols in this drink can influence enzymes that metabolize estrogens, known causal factors in breast cancer etiology.
Methods and results
We examined the associations of green tea intake (<1 time/week, 1-6 times weekly, or 7+ times weekly) with urinary estrogens and estrogen metabolites (jointly EM) in a cross-sectional sample of healthy Japanese American women, including 119 premenopausal women in luteal phase and 72 postmenopausal women. We fit robust regression models to each log-transformed EM concentration (picomoles per mg creatinine), adjusting for age and study center. In premenopausal women, intake of green tea was associated with lower luteal total EM (P trend = 0.01) and lower urinary 16-pathway EM (P trend = 0.01). In postmenopausal women, urinary estrone and estradiol were approximately 20% and 40% lower (P trend = 0.01 and 0.05, respectively) in women drinking green tea daily compared to those drinking <1 time/week. Adjustment for potential confounders (age at menarche, parity/age at first birth, body mass index, Asian birthplace, soy) did not change these associations.
Findings suggest that intake of green tea may modify estrogen metabolism or conjugation and in this way may influence breast cancer risk.
Estrogens; Metabolism; Green tea; Camellia sinensis; Breast neoplasms; Risk factors; Human; Female; Middle-aged
Endogenous estrogens and estrogen metabolism are hypothesized to be associated with premenopausal breast cancer risk but evidence is limited. We examined 15 urinary estrogens/estrogen metabolites (EM) and breast cancer risk among premenopausal women in a case-control study nested within the Nurses’ Health Study II (NHSII). In 1996–1999, urine was collected from 18,521 women during the mid-luteal menstrual phase. Breast cancer cases (N=247) diagnosed between collection and June 2005 were matched to 2 controls each (N=485). Urinary EM were measured by liquid chromatography-tandem mass spectrometry and adjusted for creatinine level. Relative risks (RRs) and 95% confidence intervals (CIs) were estimated by multivariate conditional logistic regression. Higher urinary estrone and estradiol levels were strongly significantly associated with lower risk (top vs. bottom quartile RR estrone=0.52, 95% CI=(0.30–0.88); estradiol=0.51, 95% CI=(0.30–0.86)). Generally inverse, though non-significant, patterns also were observed with 2- and 4-hydroxylation pathway EM. Inverse associations generally were not observed with 16-pathway EM and a significant positive association was observed with 17-epiestriol (top vs. bottom quartile RR=1.74, 95% CI=(1.08–2.81), p-trend=0.01). In addition, there was a significant increased risk with higher 16-pathway/parent EM ratio (comparable RR=1.61, 95% CI=(0.99–2.62), p-trend=0.04). Other pathway ratios were not significantly associated with risk except parent EM/non-parent EM (comparable RR=0.58, 95% CI=(0.35–0.96), p-trend=0.03). These data suggest that most mid-luteal urinary EM concentrations are not positively associated with breast cancer risk among premenopausal women. The inverse associations with parent EM and the parent EM/non-parent EM ratio suggest that women with higher urinary excretion of parent estrogens are at lower risk.
High systemic estrogen levels contribute to breast cancer risk for postmenopausal women, whereas low levels contribute to osteoporosis risk. Except for obesity, determinants of non-ovarian systemic estrogen levels are undefined. We sought to identify members and functions of the intestinal microbial community associated with estrogen levels via enterohepatic recirculation.
Fifty-one epidemiologists at the National Institutes of Health, including 25 men, 7 postmenopausal women, and 19 premenopausal women, provided urine and aliquots of feces, using methods proven to yield accurate and reproducible results. Estradiol, estrone, 13 estrogen metabolites (EM), and their sum (total estrogens) were quantified in urine and feces by liquid chromatography/tandem mass spectrometry. In feces, β-glucuronidase and β-glucosidase activities were determined by realtime kinetics, and microbiome diversity and taxonomy were estimated by pyrosequencing 16S rRNA amplicons. Pearson correlations were computed for each loge estrogen level, loge enzymatic activity level, and microbiome alpha diversity estimate. For the 55 taxa with mean relative abundance of at least 0.1%, ordinal levels were created [zero, low (below median of detected sequences), high] and compared to loge estrogens, β-glucuronidase and β-glucosidase enzymatic activity levels by linear regression. Significance was based on two-sided tests with α=0.05.
In men and postmenopausal women, levels of total urinary estrogens (as well as most individual EM) were very strongly and directly associated with all measures of fecal microbiome richness and alpha diversity (R≥0.50, P≤0.003). These non-ovarian systemic estrogens also were strongly and significantly associated with fecal Clostridia taxa, including non-Clostridiales and three genera in the Ruminococcaceae family (R=0.57−0.70, P=0.03−0.002). Estrone, but not other EM, in urine correlated significantly with functional activity of fecal β-glucuronidase (R=0.36, P=0.04). In contrast, fecal β-glucuronidase correlated inversely with fecal total estrogens, both conjugated and deconjugated (R≤-0.47, P≤0.01). Premenopausal female estrogen levels, which were collected across menstrual cycles and thus highly variable, were completely unrelated to fecal microbiome and enzyme parameters (P≥0.6).
Intestinal microbial richness and functions, including but not limited to β-glucuronidase, influence levels of non-ovarian estrogens via enterohepatic circulation. Thus, the gut microbial community likely affects the risk for estrogen-related conditions in older adults. Understanding how Clostridia taxa relate to systemic estrogens may identify targets for interventions.
Microbiome; Feces; Enterohepatic circulation; β-glucuronidase; β-glucosidase; Postmenopausal estrogens; Fecal estrogens; Estrogen metabolites
Photopolymerizable phospholipid DC8,9PC (1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) exhibits unique assembly characteristics in the lipid bilayer. Due to the presence of the diacetylene groups, DC8,9PC undergoes polymerization upon UV (254 nm) exposure and assumes chromogenic properties. DC8,9PC photopolymerization in a gel phase matrix lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monitored by UV-VIS absorption spectroscopy occurred within 2 minutes after UV treatment, whereas no spectral shifts were observed when DC8,9PC was incorporated in a liquid phase matrix 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Liquid chromatography-tandem mass spectrometry analysis showed a decrease in DC8,9PC monomer in both DPPC and POPC environments without any change in matrix lipids in UV-treated samples. Molecular Dynamics (MD) simulations of DPPC/DC8,9PC and POPC/DC8,9PC bilayers indicate that the DC8,9PC molecules adjust to the thickness of the matrix lipid bilayer. Furthermore, motions of DC8,9PC in the gel phase bilayer are more restricted than in the fluid bilayer. The restricted motional flexibility of DC8,9PC (in the gel phase) enables the reactive diacetylenes in individual molecules to align and undergo polymerization, whereas the unrestricted motions in the fluid bilayer restrict polymerization due to the lack of appropriate alignment of the DC8,9PC fatty acyl chains. Fluorescence microscopy data indicates homogenous distribution of the lipid probe 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (N-Rh-PE) in POPC/DC8,9PC monolayers, but domain formation in DPPC/DC8,9PC monolayers. These results show that the DC8,9PC molecules cluster and assume the preferred conformation in the gel phase matrix for UV-triggered polymerization reaction.
polymerizable lipids; lipid packing; triggered drug release; diacetylene phospholipids; light-sensitive liposomes; lipid modification; phase separation
Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44+ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.
Isothiocyanates (ITCs), such as phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), are effective cancer chemopreventive compounds. It is believed that a major mechanism for the cancer preventive activity of ITCs is through induction of cell cycle arrest and apoptosis. However, the upstream molecular targets of ITCs have been underexplored until recently. To identify proteins that are covalently modified by ITCs, human non-small cell lung cancer A549 cells were treated with 14C-PEITC and 14C-SFN and the cell lysates were extracted for analysis by 2-D gel electrophoresis and mass spectrometry. After superimposing the colloidal Coomassie blue protein staining pattern with the pattern of radioactivity obtained from X-ray films, it was clear that only a small fraction of cellular proteins contained radioactivity, presumably resulting from selective binding with PEITC or SFN via thiocarbamation. More than 30 proteins with a variety of biological functions were identified with high confidence. Here we report the identities of these potential ITC target proteins and discuss their biological relevance. The discovery of the protein targets may facilitate studies of the mechanisms by which ITCs exert their cancer preventive activity and provide molecular basis for designing more efficacious ITC compounds.
Differential 18O/16O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H218O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs, has made trypsin-catalyzed 18O post-digestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed 18O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the 18O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme-substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual 18O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then 18O/16O ratios derived via evolutionary programming. The algorithm is tested using trypsin–catalyzed 18O post-digestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both, accuracy and precision are improved utilizing this rigorous mathematical approach. Utilizing this algorithmic technique, we demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios for differentially labeled BSA peptides, by accounting for artifacts caused by a variable degree of post-digestion 18O exchange. We further demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its non myristylated mutant.
quantitation; 18O/16O stable isotope labeling; variable/incomplete 18O exchange
Members of sirtuin family regulate multiple critical biological processes, yet their role in carcinogenesis remains controversial. To investigate the physiological functions of SIRT2 in development and tumorigenesis, we disrupted Sirt2 in mice. We demonstrated that SIRT2 regulates the anaphase-promoting complex/cyclosome activity through deacetylation of its co-activators, APCCDH1 and CDC20. SIRT2 deficiency caused increased levels of mitotic regulators, including Aurora-A and -B that direct centrosome amplification, aneuploidy, and mitotic cell death. Sirt2-deficient mice develop gender-specific tumorigenesis, with females primarily developing mammary tumors, and males developing more hepatocellular carcinoma (HCC). Human breast cancers and HCC samples exhibited reduced SIRT2 levels compared with normal tissues. These data demonstrate that SIRT2 is a tumor suppressor through its role in regulating mitosis and genome integrity.
APC/C-CDH1; APC/C-CDC20; Aurora-A; mitosis; centrosome
There is mounting evidence that tumors are initiated by a rare subset of cells called cancer stem cells (CSCs). CSCs are generally quiescent, self-renew, form tumors at low numbers, and give rise to the heterogeneous cell types found within a tumor. CSCs isolated from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Here we show that vitronectin is the component of human serum driving stem cell differentiation through an integrin αVβ3-dependent mechanism. CSCs cultured on vitronectin result in downregulation of stem cell genes, modulation of differentiation markers, and loss of β-catenin nuclear localization. Blocking integrin αVβ3 inhibits differentiation and subsequently tumor formation. Thus, CSCs must be engaged by one or more extracellular signals to differentiate and initiate tumor formation, defining a new axis for future novel therapies aimed at both the extrinsic and intracellular pathways.
Prostate cancer; Breast cancer; Tumor-initiating; Vitronectin; Arginine-glycine-aspartic acid peptide; Integrin alphaVbeta3
Selective estrogen receptor modulators (SERMs) demonstrate differential endometrial cancer (EC) risk. While tamoxifen (TAM) use increases the risk of endometrial hyperplasia and malignancy, raloxifene (RAL) has neutral effects on the uterus. How TAM increases the risk of EC and why TAM and RAL differentially modulate the risk for EC, however, remain elusive. Here, we tested the hypothesis that TAM increases the risk for EC, at least in part, by enhancing the local estrogen biosynthesis and directing estrogen metabolism towards the formation of genotoxic and hormonally active estrogen metabolites. In addition, the differential effects of TAM and RAL in EC risk are attributed to their differential effect on estrogen metabolism/metabolites. The endometrial cancer cell line (Ishikawa cells) and the non-malignant immortalized human endometrial glandular cell line (EM1) were used for the study. The profile of estrogen/estrogen metabolites (EM), depurinating estrogen-DNA adducts, and the expression of estrogen-metabolizing enzymes in cells treated with 17β-estradiol (E2) alone or in combination with TAM or RAL were investigated using high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS2), ultraperformance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS), and Western blot analysis, respectively. TAM significantly increased the total EM and enhanced the formation of hormonally active and carcinogenic estrogen metabolites, 4-hydroxestrone (4-OHE1) and 16α-hydroxyestrone, with concomitant reduction in the formation of antiestrogenic and anticarcinogenic 2-hydroxyestradiol and 2-methoxyestradiol. Furthermore, TAM increased the formation of depurinating estrogen-DNA adducts 4-OHE1 -1-N7Guanine and 4-OHE1 -1-N3 Adenine. TAM-induced alteration in EM and depurinating DNA adduct formation is associated with altered expression of estrogen metabolizing enzymes CYP1A1, CYP1B1, COMT, NQO1, and SF-1 as revealed by Western blot analysis. In contrast to TAM, RAL has minimal effect on EM, estrogen-DNA adduct formation, or estrogen-metabolizing enzymes expression. These data show that TAM perturbs the balance of estrogen-metabolizing enzymes and alters the disposition of estrogen metabolites, which can explain, at least in part, the mechanism for TAM-induced EC. These results also implicate the differential effect of TAM and RAL on estrogen metabolism/metabolites as a potential mechanism for their disparate effects on the endometrium.
Endometrial cancer; Estrogen metabolism; Tamoxifen; Raloxifene
Physiological oxidants that are generated by activated phagocytes comprise the main source of oxidative stress during inflammation1,2. Oxidants such as taurine chloramine (TnCl) and hydrogen peroxide (H2O2) can damage proteins and induce apoptosis, but the role of specific protein oxidation in this process has not been defined. We found that the actin-binding protein cofilin is a key target of oxidation. When oxidation of this single regulatory protein is prevented, oxidant-induced apoptosis is inhibited. Oxidation of cofilin causes it to lose its affinity for actin and to translocate to the mitochondria, where it induces swelling and cytochrome c release by mediating opening of the permeability transition pore (PTP). This occurs independently of Bax activation and requires both oxidation of cofilin Cys residues and dephosphorylation at Ser 3. Knockdown of endogenous cofilin using targeted siRNA inhibits oxidant-induced apoptosis, which is restored by re-expression of wild-type cofilin but not by cofilin containing Cys to Ala mutations. Exposure of cofilin to TnCl results in intramolecular disulphide bonding and oxidation of Met residues to Met sulphoxide, but only Cys oxidation causes cofilin to induce mitochondrial damage.
Intake of cruciferous vegetable is inversely associated with the risk of several cancer types. Isothiocyanates (ITCs) are believed to be important constituents contributing to these cancer-preventive effects. Although several mechanisms, including induction of apoptosis, have been proposed for the anti-carcinogenesis activities of ITCs, detailed upstream triggering events are still not fully understood. Identification of ITC binding targets in cellular proteins is crucial for not only mechanistic studies but also future drug screening and design. In this review, we summarize recent progress in discovery of ITC protein targets from a technical perspective. The advantages and limitations of each method are discussed to facilitate future studies on target discovery of ITCs and perhaps other compounds.
isothiocyanates; proteomics; mass spectrometry; target identification; covalent modification
Endogenous estrogens and estrogen metabolites play an important role in the pathogenesis and development of human breast, endometrial, and ovarian cancers. Increasing evidence also supports their involvement in the development of certain lung, colon and prostate cancers.
In this study we systemically surveyed endogenous estrogen and estrogen metabolite levels in each of the NCI-60 human tumor cell lines, which include human breast, central nerve system, colon, ovarian, prostate, kidney and non-small cell lung cancers, as well as melanomas and leukemia. The absolute abundances of these metabolites were measured using a liquid chromatography-tandem mass spectrometry method that has been previously utilized for biological fluids such as serum and urine.
Endogenous estrogens and estrogen metabolites were found in all NCI-60 human tumor cell lines and some were substantially elevated and exceeded the levels found in well known estrogen-dependent and estrogen receptor-positive tumor cells such as MCF-7 and T-47D. While estrogens were expected to be present at high levels in cell lines representing the female reproductive system (that is, breast and ovarian), other cell lines, such as leukemia and colon, also contained very high levels of these steroid hormones. The leukemia cell line RMPI-8226 contained the highest levels of estrone (182.06 pg/106 cells) and 17β-estradiol (753.45 pg/106 cells). In comparison, the ovarian cancer cell line with the highest levels of these estrogens contained only 19.79 and 139.32 pg/106 cells of estrone and 17β-estradiol, respectively. The highest levels of estrone and 17β-estradiol in breast cancer cell lines were only 8.45 and 87.37 pg/106 cells in BT-549 and T-47D cells, respectively.
The data provided evidence for the presence of significant amounts of endogenous estrogens and estrogen metabolites in cell lines not commonly associated with these steroid hormones. This broad discovery of endogenous estrogens and estrogen metabolites in these cell lines suggest that several human tumors may be beneficially treated using endocrine therapy aimed at estrogen biosynthesis and estrogen-related signaling pathways.
It is well-established that 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) causes acute liver damage in animals and humans. The aim of this study was to identify and characterize oxidative modification and inactivation of cytosolic proteins in MDMA-exposed rats. Markedly increased levels of oxidized and nitrated cytosolic proteins were detected 12 h after the second administration of two consecutive MDMA doses (10 mg/kg each). Comparative two dimensional gel electrophoresis (2-DE) analysis showed markedly increased levels of biotin-N-methylimide (biotin-NM)-labeled oxidized cytosolic proteins in MDMA-exposed rats compared to vehicle-treated rats. Proteins in the 22 gel spots of strong intensities were identified using tandem mass spectrometry (MS/MS). The oxidatively-modified proteins identified include antioxidant defensive enzymes, a calcium-binding protein, and proteins involved in metabolism of lipids, nitrogen, and carbohydrates (glycolysis). Cytosolic superoxide dismutase was oxidized and its activity significantly inhibited following MDMA exposure. Consistent with the oxidative inactivation of peroxiredoxin, MDMA activated c-Jun N-terminal protein kinase and p38 kinase. Since these protein kinases phosphorylate anti-apoptotic Bcl-2 protein, their activation may promote apoptosis in MDMA-exposed tissues. Our results show for the first time that MDMA induces oxidative-modification of many cytosolic proteins accompanied with increased oxidative stress and apoptosis, contributing to hepatic damage.
Cytosolic proteins; liver damage; MDMA; oxidative-modification; redox-based proteomics
The high incidence of and few identified risk factors for prostate cancer underscore the need to further evaluate markers of prostate carcinogenesis. The aim of this pilot study was to evaluate urinary estrogen metabolites as a biomarker of prostate cancer risk.
Using a liquid chromatography-tandem mass spectrometry method, urinary concentrations of 15 estrogen metabolites were determined in 77 prostate cancer cases, 77 healthy controls, and 37 subjects who had no evidence of prostate cancer after a prostate biopsy.
We observed an inverse association between the urinary 16-ketoestradiol (16-KE2) and 17-epiestriol (17-epiE3)- metabolites with high estrogenic activity- and prostate cancer risk. Men in the lowest quartile of 16-KE2, had a 4.6-fold risk of prostate cancer (OR= 4.62, 95% CI =1.34–15.99), compared with those in the highest quartile.
We observed modest differences in estrogen metabolite concentrations between prostate cancer patients and subjects without cancer. Larger studies with both androgen and estrogen measurements are needed to confirm these results to clarify further whether estrogen metabolites are independent biomarkers for prostate cancer risk and whether androgen/estrogen imbalance influences prostate cancer risk.
prostate cancer; estrogen metabolites; benign prostatic hyperplasia; case-control study
We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1). The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis.
Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five non-cysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.
Quantitative proteomics; combined 16O/18O and ICAT stable isotopic labeling; Triton X-100 and Brij-96 detergent-insoluble membrane proteins
Described is a method that relies on subtractive tissue-directed shot-gun proteomics to identify tumor proteins in the blood of a patient newly diagnosed with cancer. To avoid analytical and statistical biases caused by physiologic variability of protein expression in the human population, this method was applied on clinical specimens obtained from a single patient diagnosed with non-metastatic renal cell carcinoma (RCC). The proteomes extracted from tumor, normal adjacent tissue and pre-operative plasma were analyzed using 2D-LC-MS. The lists of identified proteins were filtered to discover proteins that i) were found in tumor but not normal tissue, ii) were identified in matching plasma, and iii) whose spectral count was higher in tumor tissue than plasma. These filtering criteria resulted in identification of eight tumor proteins in the blood. Subsequent Western-blot analysis confirmed the presence of cadherin-5, cadherin-11, DEAD-box protein-23, and pyruvate kinase) in the blood of the patient under the study, as well as in the blood of four other patients diagnosed with RCC. These results demonstrate the utility of a combined blood/tissue analysis strategy that permits the detection of tumor proteins in the blood of a patient diagnosed with RCC.
In preparation for large-scale epidemiologic studies of the role of estrogen metabolism in the etiology of breast and other cancers, we examined the stability of estrogens and estrogen metabolites (EM) in urine during processing and storage protocols.
Fifteen EM were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in first morning urines from three premenopausal women. Linear regression was used to model log EM concentrations for each woman, with and without adding ascorbic acid (0.1% w/v), during storage at 4 °C (7–8 time-points, up to 48 h), during long-term storage at −80 °C (10 time-points, up to 1 y) and by freeze-thaw cycles (up to 3).
Without ascorbic acid, concentrations (pmol/mL) of nearly all EM changed <1% per 24 h of storage at 4 °C, and <1% during storage at −80 °C for one year; similarly, thawing and refreezing samples three times was not consistently associated with losses for any EM. Ascorbic acid had no clear beneficial effect on EM stability in these experiments.
Given the large inter-individual variability in urinary EM concentrations, changes of the magnitude observed here are unlikely to cause substantial misclassification. Furthermore, processing and storage conditions studied here are adequate for use in epidemiologic studies.
estradiol; estrogens; estrogen metabolites; stability; urine
Small Heterodimer Partner (SHP) inhibits numerous transcription factors that are involved in diverse biological processes, including lipid and glucose metabolism. In response to increased hepatic bile acids, SHP gene expression is induced and the SHP protein is stabilized. We now show that the activity of SHP is also increased by posttranslational methylation at Arg-57 by protein arginine methyltransferase 5 (PRMT5). Adenovirus-mediated hepatic depletion of PRMT5 decreased SHP methylation and reversed the suppression of metabolic genes by SHP. Mutation of Arg-57 decreased SHP interaction with its known cofactors, Brm, mSin3A, and histone deacetylase 1 (HDAC1), but not with G9a, and decreased their recruitment to SHP target genes in mice. Hepatic overexpression of SHP inhibited metabolic target genes, decreased bile acid and hepatic triglyceride levels, and increased glucose tolerance. In contrast, mutation of Arg-57 selectively reversed the inhibition of SHP target genes and metabolic outcomes. The importance of Arg-57 methylation for the repression activity of SHP provides a molecular basis for the observation that a natural mutation of Arg-57 in humans is associated with the metabolic syndrome. Targeting posttranslational modifications of SHP may be an effective therapeutic strategy by controlling selected groups of genes to treat SHP-related human diseases, such as metabolic syndrome, cancer, and infertility.
Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate) (PPS), iii) a combination of both agents (methanol + PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was ~98% positive for CD14 expression by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46 %) were mapped integral membrane proteins. However, employing PPS a total of 216 proteins of which 75 (35 %) were mapped integral membrane proteins. These results indicate that methanol unaided or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.
CD14 monocyte; Membrane proteins; Solubilization; Methanol; Detergents; LC-MS/MS
Sphingomyelin (SM) and ceramide-phosphoethanolamines (cer-PE) are related lipids present in mammals and insects, respectively. Owing to the critical roles that cer-PE play in eukaryotic cellular function, there is a need to develop methods that provide accurate quantitation of these compounds. Results obtained in this study demonstrate that Drosophila contains cer-PE’s with unsaturated sphingoid base cores as well as low levels of cer-PE’s that possess saturated sphingoid base cores. Specifically, the method developed in this study enabled the quantitation of picogram amounts of cer-PE containing both unsaturated d14:1Δ4 and d16:1Δ4 and saturated d14:0 sphingoid base cores. Using this method cer-PE compounds with both saturated and unsaturated sphingoid base core were initially identified by neutral loss scanning, followed by quantitation using single reaction monitor scans (SRM). The SRM scans measured a product ion originating from the sphingoid base backbone, rather than from the head group, increasing the specificity and the sensitivity of the quantation measurement.
Drosophila; Liquid chromatography; Neutral loss-single ion monitoring scans; Selected reaction monitoring; Quantitation; Ceramide-phosphorylethanolamines; Sphingoid bases
Neuroblastoma (NB) is a heterogeneous pediatric tumor. To better understand the biological pathways involved in the development of high-risk neuroblastoma, we performed parallel global protein and mRNA expression profiling on NB tumors of stage4 MYCN-amplified (4+) and stage1 MYCN-not-amplified (1-) using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray respectively. A total of 1461 proteins represented by 2 or more peptides were identified from the quantitative ICAT analysis, of which 433 and 130 proteins are up- or down-regulated respectively in 4+ tumor compared to the 1- tumor. Pathway analysis of the differentially expressed proteins showed the enrichment of glycolysis, DNA replication and cell cycle processes in the up-regulated proteins and cell adhesion, nervous system development and cell differentiation processes in the down-regulated proteins in 4+ tumor; suggesting a less mature neural and a more invasive phenotype of 4+ tumor. Myc targets and ribosomal proteins are over represented in the 4+ tumors as expected; functional gene sets reported to be enriched in neural and embryonic stem cells are significantly enriched in the 4+ tumor, indicating the existence of a stemness signature in MYCN-amplified stage 4 tumor. In addition, protein and mRNA expression are moderately correlated (r = 0.51, p < 0.0001), as approximately half of the up-regulated proteins in 4+ tumor have elevated mRNA level (n=208), and 1/3 of down regulated proteins have lower mRNA expression (n=47). Further biological network analysis revealed that the differentially expressed proteins closely interact with other proteins of known networks; the important role of MYCN is confirmed and other transcription factors identified in the network may have potential roles in the biology of NB tumor. We used global genomic and proteomic analysis to identify biologically relevant proteins and pathways important to NB progression and development that may provide new insights into the biology of advanced neuroblastoma.
neuroblastoma; ICAT; pathway analysis; proteomics; genomics
Absolute and relative concentrations of estrogens and estrogen metabolites (EM) are important for clinical decisions, as well as for epidemiologic, experimental, and clinical research on hormonal carcinogenesis. Radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) are routinely used for measuring EM in blood and urine due to efficiency and low cost. Here we compare absolute and ranked concentrations of estrone, estradiol, and estriol measured by indirect RIA and of 2-hydroxyestrone and 16α-hydroxyestrone measured by ELISA to the concentrations obtained using a novel liquid-chromatography-tandem mass spectrometry (LC-MS/MS) method which measures 15 EM concurrently. We used overnight urine samples collected from control women (362 premenopausal, 168 postmenopausal) participating in a population-based case-control study of breast cancer among Asian-American women ages 20–55 years. When comparing RIA or ELISA levels to LC-MS/MS, absolute concentrations for the five EM ranged from 1.6–2.9 and 1.4–11.8 times higher in premenopausal and postmenopausal women, respectively, (all p<0.0001). However, LC-MS/MS measurements were highly correlated [Spearman r (rs) =0.8–0.9] with RIA and ELISA measurements in premenopausal women, and moderately correlated (rs=0.4–0.8) in postmenopausal women. Measurements of the 2-hydroxyestrone:16α-hydroxyestrone ratio, a putative biomarker of breast cancer risk, were moderately correlated in premenopausal women (rs=0.6–0.7) but only weakly correlated in postmenopausal women (rs=0.2). LC-MS/MS had higher intraclass correlation coefficients (≥99.6%) and lower coefficients of variation (≤9.4%) than ELISA (≥97.2% and ≤14.2%) and RIA (≥95.2% and ≤17.8%). Comparison with the LC-MS/MS method suggests that the widely used RIA and ELISA EM measures may be problematic, especially at low EM levels characteristic of postmenopausal women.
estrone; estradiol; LC-MS; RIA; ELISA