ABSTRACT
PURPOSE: Ewing sarcoma is the second most common bone sarcoma in children, with 1 case per 1.5 million in the United States. Although the survival rate of patients diagnosed with localized disease is approximately 70%, this decreases to approximately 30% for patients with metastatic disease and only approximately 10% for treatment-refractory disease, which have not changed for decades. Therefore, new therapeutic strategies are urgently needed for metastatic and refractory Ewing sarcoma. EXPERIMENTAL DESIGN: This study analyzed 19 unique Ewing sarcoma patient- or cell line-derived xenografts (from 14 primary and 5 metastatic specimens) using proteomics to identify surface proteins for potential immunotherapeutic targeting. Plasma membranes were enriched using density gradient ultracentrifugation and compared with a reference standard of 12 immortalized non-Ewing sarcoma cell lines prepared in a similar manner. In parallel, global proteome analysis was carried out on each model to complement the surfaceome data. All models were analyzed by Tandem Mass Tags-based mass spectrometry to quantify identified proteins. RESULTS: The surfaceome and global proteome analyses identified 1,131 and 1,030 annotated surface proteins, respectively. Among surface proteins identified, both approaches identified known Ewing sarcoma-associated proteins, including IL1RAP, CD99, STEAP1, and ADGRG2, and many new cell surface targets, including ENPP1 and CDH11. Robust staining of ENPP1 was demonstrated in Ewing sarcoma tumors compared with other childhood sarcomas and normal tissues. CONCLUSIONS: Our comprehensive proteomic characterization of the Ewing sarcoma surfaceome provides a rich resource of surface-expressed proteins in Ewing sarcoma. This dataset provides the preclinical justification for exploration of targets such as ENPP1 for potential immunotherapeutic application in Ewing sarcoma. See related commentary by Bailey, p. 934.
Subject(s)
Bone Neoplasms , Sarcoma, Ewing , Sarcoma , Child , Humans , Sarcoma, Ewing/genetics , Sarcoma, Ewing/therapy , Membrane Proteins , Proteome , Proteomics , Bone Neoplasms/genetics , Bone Neoplasms/therapy , Immunotherapy , Antigens, Neoplasm , OxidoreductasesABSTRACT
BACKGROUND: Androgen deprivation therapy (ADT) is the standard of care for prostate cancer treatment. Studies suggest that patients with testosterone levels below 0.7 nM have a longer time to castration resistance. Using the most accurate testosterone measurement method, namely mass spectrometry (MS), we sought to determine if a lower testosterone level under ADT could be associated with longer time to castration resistance. METHODS: This retrospective study included 138 prostate cancer patients undergoing noncurative continuous ADT for which we had access to testosterone measurements assessed by MS. For 108 samples, paired immunoassays (IA) testosterone measurement was available. Primary outcome was time to castration-resistant prostate cancer (CRPC). The Contal and O'Quigley method was used to determine the optimal testosterone castration cut-off point considering the outcome and time-to-event variables. Relationship between testosterone levels assessed either by IA or MS and time to CRPC was evaluated using Cox regression. RESULTS: Mean testosterone level was 0.370 nM by IA and 0.275 nM as assessed by MS. The optimal testosterone cut-off point identified to predict time to CRPC was of 0.705 nM for IA and of 0.270 nM for MS. While no significant difference for time to CRPC was found between patients showing IA testosterone level ≥0.705 nM versus <0.705 nM (hazard ratio [HR]: 1.579; 95% confidence interval [CI]: 0.908-2.745), patients with MS testosterone ≥0.270 nM had an increased risk of progression to CRPC compared to MS testosterone <0.270 nM in univariate (HR: 1.717; 95% CI: 1.160-2.541) and multivariate analysis (HR: 1.662; 95% CI: 1.043-2.648). CONCLUSIONS: The higher sensitivity of MS testosterone measurement methods allows the identification of a lower castration threshold and leads to early identification of patients more likely to progress to CRPC. These patients would likely benefit from treatment intensification by androgen receptor axis-targeted therapies to delay disease progression.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Prostatic Neoplasms , Male , Humans , Prostatic Neoplasms/drug therapy , Testosterone , Androgen Antagonists/therapeutic use , Androgens , Prostatic Neoplasms, Castration-Resistant/drug therapy , Retrospective Studies , OrchiectomyABSTRACT
PURPOSE: Dihydrotestosterone and testosterone are thought to be major contributors of prostate cancer progression and resistance. We studied the modulation of 15 circulating steroids by castration and their association with dihydrotestosterone and testosterone levels. MATERIALS METHODS: A total of 116 serum samples were collected from 99 prostate cancer patients and categorized as eugonadal, castration-sensitive prostate cancer, castration-resistant prostate cancer, or castration-resistant prostate cancer under abiraterone acetate. Serum levels of 15 steroids were measured using mass spectrometry and compared between groups using analysis of variance. Intrapatient association of steroid levels and the androgens testosterone and dihydrotestosterone were assessed using Pearson correlation and linear regression. RESULTS: Testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androsterone, androstenediol, estrone, estrone-sulfate, estradiol, and androsterone/3α-diol-3/3α-diol-17-glucuronide levels were significantly decreased in castration-sensitive prostate cancer (castrated) compared to eugonadal patients. Testosterone levels were strongly associated with multiple steroids under eugonadal conditions, whereas they were weakly affected by precursor steroids in castrated patients. By contrast, dihydrotestosterone levels under androgen deprivation therapy were associated with testosterone and the backdoor pathway metabolite androsterone. In castration-resistant prostate cancer patients, levels of androstenedione were significantly associated with testosterone level, while testosterone was the only steroid associated with dihydrotestosterone levels. CONCLUSIONS: Androgen deprivation therapy significantly reduces the levels of 13 circulating steroids. Upon androgen deprivation therapy initiation, the backdoor pathway metabolite androsterone are strongly associated with dihydrotestosterone levels. Under castration-resistant prostate cancer conditions, androstenedione was significantly associated with testosterone levels, suggesting the presence of tumor-related circulating androgens in these patients. These results provide further rationale to intensify treatments with androgen receptor axis signaling pathway inhibitors in patients with prostate cancer.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Prostatic Neoplasms , Male , Humans , Androgens , Androstenedione/pharmacology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/surgery , Dihydrotestosterone/metabolism , Androgen Antagonists/therapeutic use , Androsterone , Prostatic Neoplasms, Castration-Resistant/drug therapy , Estrone , Testosterone , Orchiectomy , Dehydroepiandrosterone , SulfatesABSTRACT
Ewing sarcoma (EwS) is a highly aggressive cancer and the second most common malignant bone tumor of children and young adults. Although patients with localized disease have a survival rate of approximately 75%, the prognosis for patients with metastatic disease remains dismal (<30%) and has not improved in decades. Standard-of-care treatments include local therapies such as surgery and radiotherapy, in addition to poly-agent adjuvant chemotherapy, and are often associated with long-term disability and reduced quality of life. Novel targeted therapeutic strategies that are more efficacious and less toxic are therefore desperately needed, particularly for metastatic disease, given that the presence of metastasis remains the most powerful predictor of poor outcome in EwS. Intercellular communication within the tumor microenvironment is emerging as a crucial mechanism for cancer cells to establish immunosuppressive and cancer-permissive environments, potentially leading to metastasis. Altering this communication within the tumor microenvironment, thereby preventing the transfer of oncogenic signals and molecules, represents a highly promising therapeutic strategy. To achieve this, extracellular vesicles (EVs) offer a candidate mechanism as they are actively released by tumor cells and enriched with proteins and RNAs. EVs are membrane-bound particles released by normal and tumor cells, that play pivotal roles in intercellular communication, including cross-talk between tumor, stromal fibroblast, and immune cells in the local tumor microenvironment and systemic circulation. EwS EVs, including the smaller exosomes and larger microvesicles, have the potential to reprogram a diversity of cells in the tumor microenvironment, by transferring various biomolecules in a cell-specific manner. Insights into the various biomolecules packed in EwS EVs as cargos and the molecular changes they trigger in recipient cells of the tumor microenvironment will shed light on various potential targets for therapeutic intervention in EwS. This review details EwS EVs composition, their potential role in metastasis and in the reprogramming of various cells of the tumor microenvironment, and the potential for clinical intervention.
ABSTRACT
PURPOSE: Beyond testosterone, several steroids contribute to the activation of the androgen receptor pathway, but their relative contributions to the activation of the androgen receptor signaling axis in patients with castrated prostate cancer remain unknown. MATERIALS AND METHODS: Serum levels of 9 steroids were measured by mass spectrometry from continuously castrated patients of the PR.7 study (219) and from the PCA24 cohort (116). For each steroid standard curves for dose dependent prostate specific antigen promoter activation were built in castration sensitive (LAPC4) and resistant (VCaP) prostate cancer models. Standard curves were used to determine the androgen receptor activation potency for each steroid measurement from patients in these trials. RESULTS: In LAPC4 and VCaP cells testosterone, dihydrotestosterone and androstenedione induced androgen receptor transcriptional activity, while dehydroepiandrosterone, 5alpha-androstan-3beta,17beta-diol, androstenediol and androsterone stimulated androgen receptor only in VCaP cells. Extragonadal steroids were responsible for 34% (LAPC4) and 88% (VCaP) of the serum total androgen receptor transcriptional activity found in castrated cases. The total androgen receptor transcriptional activity secondary to testosterone, dihydrotestosterone and androstenedione was associated with time to castration resistance in patients from the PR.7 study (HR 2.17, 95% CI 1.12-4.23, p=0.02) in multivariate analysis using the castration sensitive model (LAPC4). Androgen receptor transcriptional activity of extragonadal androstenedione was the only steroid statistically associated with time to castration resistance in univariate analysis (HR 1.89, 95% CI 1.04-3.44, p=0.036). CONCLUSIONS: Extragonadal steroids contribute significantly to the androgen receptor axis activation at castration levels of testosterone in recurrent nonmetastatic prostate cancer and these sustain the development of castration resistance after primary local treatment.
Subject(s)
Androstenedione/pharmacology , Castration/methods , Neoplasm Recurrence, Local/therapy , Prostatic Neoplasms/therapy , Receptors, Androgen/metabolism , Testosterone/analogs & derivatives , Testosterone/pharmacology , Anabolic Agents/pharmacology , Androgens/pharmacology , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Follow-Up Studies , Humans , Male , Mass Spectrometry , Neoplasm Recurrence, Local/metabolism , Neoplasm Recurrence, Local/pathology , Prospective Studies , Prostate/drug effects , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Receptors, Androgen/drug effects , Time FactorsABSTRACT
BACKGROUND: The development of phenotypic biomarkers to aid the selection of treatment for patients with castrate-resistant prostate cancer (CRPC) is an important priority. Plasma exosomes have excellent potential as real-time biomarkers to characterize the tumor because they are easily accessible in the blood and contain DNA, RNA, and protein from the parent cell. This study aims to investigate the characteristics of putative prostate-specific plasma extracellular vesicle (EV) markers and their relationship with clinical outcomes. METHODS AND PATIENTS: We investigated plasma EVs in a total of 89 patients with prostate cancer (PCa) at different stages of disease progression. EVs were isolated using both precipitation and ultracentrifugation methods; physical characterization was performed using dynamic light scattering, acetylcholinesterase (AChE) activity, and velocity gradients. An immunocapture method was developed for the evaluation of prostate-specific membrane antigen (PSMA)-positive exosomes. Exosomal messenger RNA (mRNA) was quantified using droplet digital polymerase chain reaction for the expression of KLK3 and androgen receptor splice variant 7 (AR-V7) genes, which code prostate-specific antigen (PSA) and AR-V7, respectively. Serum sex steroids were measured using liquid chromatography-tandem mass spectroscopy. RESULTS: Isolated exosomes from patients with CRPC had a smaller hydrodynamic size than those isolated from localized patients with PCa, while AChE activity showed no difference. Moreover, no differences were observed after initiation of androgen deprivation therapy in serial patient samples. Velocity gradients identified that PSMA-positive exosomes occupied a specific fraction of isolated EVs. A total of 35 patients with CRPC had mRNA analyzed from isolated plasma exosomes. Detectable exosomal KLK3 corresponded with higher concomitant serum PSA measurements, as expected (mean, 112.6 vs 26.61 ng/mL; P = .065). Furthermore, detectable levels of AR-V7 mRNA were associated with a shorter time to progression (median, 16.0 vs 28.0 months; P = .0499). Furthermore, detectable exosomal AR-V7 was significantly associated with testosterone levels below the lower limit of quantification (<0.1 nM). CONCLUSIONS: Our results suggest that exosomal AR-V7 is correlated with lower sex steroid levels in CRPC patients with a poorer prognosis. PSMA immunocapture does not appear sufficient to isolate PCa-specific exosomes.
Subject(s)
Extracellular Vesicles/metabolism , Prostatic Neoplasms, Castration-Resistant/diagnosis , Aged , Aged, 80 and over , Biomarkers, Tumor , Disease Progression , Humans , Kallikreins/metabolism , Male , Middle Aged , Phenotype , Progression-Free Survival , Prostate-Specific Antigen/blood , Prostate-Specific Antigen/metabolism , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/mortality , Prostatic Neoplasms, Castration-Resistant/pathology , Survival RateABSTRACT
Failure to suppress testosterone below 0.7 nM in castrated prostate cancer patients is associated with poor clinical outcomes. Testosterone levels in castrated patients are therefore routinely measured. Although mass spectrometry is the gold standard used to measure testosterone, most hospitals use an immunoassay method. In this study, we sought to evaluate the accuracy of an immunoassay method to measure castrate testosterone levels, with mass spectrometry as the reference standard. We retrospectively evaluated a cohort of 435 serum samples retrieved from castrated prostate cancer patients from April to September 2017. No follow-up of clinical outcomes was performed. Serum testosterone levels were measured in the same sample using liquid chromatography coupled with tandem mass spectrometry and electrochemiluminescent immunoassay methods. The mean testosterone levels were significantly higher with immunoassay than with mass spectrometry (0.672 ± 0.359 vs 0.461 ± 0.541 nM; P < 0.0001). Half of the samples with testosterone ≥0.7 nM assessed by immunoassay were measured <0.7 nM using mass spectrometry. However, we observed that only 2.95% of the samples with testosterone <0.7 nM measured by immunoassay were quantified ≥0.7 nM using mass spectrometry. The percentage of serum samples experiencing testosterone breakthrough at >0.7 nM was significantly higher with immunoassay (22.1%) than with mass spectrometry (13.1%; P < 0.0001). Quantitative measurement of serum testosterone levels >0.7 nM by immunoassay can result in an inaccurately identified castration status. Suboptimal testosterone levels in castrated patients should be confirmed by either mass spectrometry or an immunoassay method validated at low testosterone levels and interpreted with caution before any changes are made to treatment management.
ABSTRACT
The conjugative metabolism mediated by UDP-glucuronosyltransferase enzymes (UGTs) significantly influences the bioavailability and biological responses of endogenous molecule substrates and xenobiotics including drugs. UGTs participate in the regulation of cellular homeostasis by limiting stress induced by toxic molecules, and by controlling hormonal signaling networks. Glucuronidation is highly regulated at genomic, transcriptional, post-transcriptional and post-translational levels. However, the UGT protein interaction network, which is likely to influence glucuronidation, has received little attention. We investigated the endogenous protein interactome of human UGT1A enzymes in main drug metabolizing non-malignant tissues where UGT expression is most prevalent, using an unbiased proteomics approach. Mass spectrometry analysis of affinity-purified UGT1A enzymes and associated protein complexes in liver, kidney and intestine tissues revealed an intricate interactome linking UGT1A enzymes to multiple metabolic pathways. Several proteins of pharmacological importance such as transferases (including UGT2 enzymes), transporters and dehydrogenases were identified, upholding a potential coordinated cellular response to small lipophilic molecules and drugs. Furthermore, a significant cluster of functionally related enzymes involved in fatty acid ß-oxidation, as well as in the glycolysis and glycogenolysis pathways were enriched in UGT1A enzymes complexes. Several partnerships were confirmed by co-immunoprecipitations and co-localization by confocal microscopy. An enhanced accumulation of lipid droplets in a kidney cell model overexpressing the UGT1A9 enzyme supported the presence of a functional interplay. Our work provides unprecedented evidence for a functional interaction between glucuronidation and bioenergetic metabolism.
ABSTRACT
In the fight against androgen-sensitive prostate cancer, the enzyme 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is an attractive therapeutic target considering its key role in the formation of androgenic steroids. In this study, we attempted to assess the in vivo efficacy of the compound RM-532-105, an androsterone derivative developed as an inhibitor of 17ß-HSD3, in the prostate cancer model of androgen-sensitive LAPC-4 cells xenografted in nude mice. RM-532-105 did not inhibit the tumor growth induced by 4-androstene-3,17-dione (4-dione); rather, the levels of the androgens testosterone (T) and dihydrotestosterone (DHT) increased within the tumors. In plasma, however, DHT levels increased but T levels did not. In troubleshooting experiments, the non-androgenic potential of RM-532-105 was confirmed by two different assays (LAPC-4 proliferation and androgen receptor transcriptional activity assays). The enzyme 5α-reductase was also revealed to be the predominant enzyme metabolizing 4-dione in LAPC-4 cells, yielding 5α-androstane-3,17-dione and not T. Other 17ß-HSDs than 17ß-HSD3 seem responsible in the androgen synthesis. From experiments with LAPC-4 cells, we fortuitously came across the interesting finding that 17ß-HSD3 inhibitor RM-532-105 is concentrated inside tumors.
Subject(s)
17-Hydroxysteroid Dehydrogenases/antagonists & inhibitors , Androstanes/therapeutic use , Enzyme Inhibitors/therapeutic use , Prostatic Neoplasms/drug therapy , Sulfonamides/therapeutic use , Androstanes/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Dihydrotestosterone/blood , Enzyme Inhibitors/pharmacology , Humans , Male , Mice , Mice, Nude , Sulfonamides/pharmacologyABSTRACT
Alternative splicing at the human glucuronosyltransferase 1 gene locus (UGT1) produces alternate isoforms UGT1A_i2s that control glucuronidation activity through protein-protein interactions. Here, we hypothesized that UGT1A_i2s function as a complex protein network connecting other metabolic pathways with an influence on cancer cell metabolism. This is based on a pathway enrichment analysis of proteomic data that identified several high-confidence candidate interaction proteins of UGT1A_i2 proteins in human tissues-namely, the rate-limiting enzyme of glycolysis pyruvate kinase (PKM), which plays a critical role in cancer cell metabolism and tumor growth. The partnership of UGT1A_i2 and PKM2 was confirmed by coimmunoprecipitation in the HT115 colon cancer cells and was supported by a partial colocalization of these two proteins. In support of a functional role for this partnership, depletion of UGT1A_i2 proteins in HT115 cells enforced the Warburg effect, with a higher glycolytic rate at the expense of mitochondrial respiration, and led to lactate accumulation. Untargeted metabolomics further revealed a significantly altered cellular content of 58 metabolites, including many intermediates derived from the glycolysis and tricarboxylic acid cycle pathways. These metabolic changes were associated with a greater migration potential. The potential relevance of our observations is supported by the down-regulation of UGT1A_i2 mRNA in colon tumors compared with normal tissues. Alternate UGT1A variants may thus be part of the expanding compendium of metabolic pathways involved in cancer biology directly contributing to the oncogenic phenotype of colon cancer cells. Findings uncover new aspects of UGT functions diverging from their transferase activity.
Subject(s)
Alternative Splicing/genetics , Colonic Neoplasms/enzymology , Colonic Neoplasms/metabolism , Glucuronosyltransferase/genetics , Carrier Proteins/metabolism , Cell Adhesion , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Respiration , Cell Survival , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Energy Metabolism , Gene Expression Regulation, Neoplastic , Glucuronosyltransferase/metabolism , Glycolysis , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Lactic Acid/metabolism , Membrane Proteins/metabolism , Metabolomics , Mitochondria/metabolism , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Thyroid Hormones/metabolism , Thyroid Hormone-Binding ProteinsABSTRACT
PURPOSE: Reliable biomarkers that predict prostate cancer outcomes are urgently needed to improve and personalize treatment approaches. With this goal in mind, we individually and collectively appraised common genetic polymorphisms related to estradiol metabolic pathways to find prostate cancer prognostic markers. METHODS: The genetic profiles of 526 men with organ-confined prostate cancer were examined to find common genetic polymorphisms related to estradiol metabolic pathways and these findings were replicated in a cohort of 213 men with more advanced disease (follow-up time for both cohorts, >7.4 years). Specifically, we examined 71 single-nucleotide polymorphisms (SNP) in SULT2A1, SULT2B1, CYP1B1, COMT, CYP3A4, CYP3A5, CYP3A43, NQO1, and NQO2 and assessed the impact of the SNPs alone and in combination on prostate cancer progression and on circulating hormone levels. RESULTS: According to a multivariate analysis, CYP1B1 (rs1800440), COMT (rs16982844), and SULT2B1 (rs12460535, rs2665582, rs10426628) were significantly associated with prostate cancer progression and hormone levels. Remarkably, by combining the SNP information with previously identified HSD17B2 markers, the patients could be stratified into four distinct prognostic subgroups. The most prominent association was observed for the eight-marker combination [CYP1B1 (rs1800440), SULT2B1 (rs12460535, rs2665582, and rs10426628), and HSD17B2 (rs4243229, rs1364287, rs2955162, and rs1119933)]. CONCLUSION: This study identified specific germline variations in estradiol metabolism-related pathways, namely CYP1B1, SULT2B1, and HSD17B2, as novel prognostic markers that are cumulatively associated with increased risk of prostate cancer progression. This panel of markers warrants additional investigation and validation to help stratify patients according to their risk of progression. Clin Cancer Res; 20(11); 2971-83. ©2014 AACR.
Subject(s)
Cytochrome P-450 CYP1B1/genetics , Estradiol Dehydrogenases/genetics , Estradiol/metabolism , Polymorphism, Single Nucleotide , Prostatic Neoplasms/genetics , Sulfotransferases/genetics , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Disease Progression , Estradiol/genetics , Genetic Predisposition to Disease , Gonadal Steroid Hormones/blood , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Prognosis , Prostatic Neoplasms/mortality , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
PURPOSE: Polymorphisms in the genes SRD5A1 and SRD5A2 encoding androgen biosynthetic 5α-reductase enzymes have been associated with an altered risk of biochemical recurrence after radical prostatectomy in localized prostate cancer. EXPERIMENTAL DESIGN: To gain potential insights into SRD5A biologic effects, we examined the relationship between SRD5A prognostic markers and endogenous sex-steroid levels measured by mass spectrometry in plasma samples and corresponding prostatic tissues of patients with prostate cancer. RESULTS: We report that five of the seven SRD5A markers differentially affect sex-steroid profiles of dihydrotestosterone and its metabolites in both the circulation and prostatic tissues of patients with prostate cancer. Remarkably, a 32% increase in intraprostatic testosterone levels was observed in the presence of the high-risk SRD5A rs2208532 polymorphism. Moreover, SRD5A2 markers were associated predominantly with circulating levels of inactive glucuronides. Indeed, the rs12470143 SRD5A2 protective allele was associated with high circulating androstane-3α, 17ß-diol-17-glucuronide (3α-diol-17G) levels as opposed to lower levels of both 3α-diol-17G and androsterone-glucuronide observed with the rs2208532 SRD5A2 risk allele. Moreover, SRD5A2 rs676033 and rs523349 (V89L) risk variants, in strong linkage disequilibrium, were associated with higher circulating levels of 3α-diol-3G. The SRD5A2 rs676033 variant further correlated with enhanced intraprostatic exposure to 5α-reduced steroids (dihydrotestosterone and its metabolite 3ß-diol). Similarly, the SRD5A1 rs166050C risk variant was associated with greater prostatic exposure to androsterone, whereas no association was noted with circulating steroids. CONCLUSIONS: Our data support the association of 5α-reductase germline polymorphisms with the hormonal milieu in patients with prostate cancer. Further studies are needed to evaluate if these variants influence 5α-reductase inhibitor efficacy.
Subject(s)
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics , Androgens/genetics , Membrane Proteins/genetics , Polymorphism, Single Nucleotide , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Adult , Aged , Aged, 80 and over , Androgens/metabolism , Genotype , Humans , Male , Mass Spectrometry , Middle AgedABSTRACT
Transcripts of the UGT1A gene, encoding half of human UDP-glucuronosyltransferase (UGT) enzymes, undergo alternative splicing, resulting in active enzymes named isoforms 1 (i1s) and novel truncated isoforms 2 (i2s). Here, we investigated the effects of depleting endogenous i2 on drug response and attempted to unveil any additional biologic role(s) for the truncated novel UGT proteins. We used an integrated systems biology approach that combines RNA interference with unbiased global genomic and proteomic screens, and used HT115 colorectal cancer cells as a model. Consistent with previous evidence suggesting that i2s negatively regulate i1s through protein-protein interactions, i2-depleted cells were less sensitive to drug-induced cell death (IC50 of 0.45 ± 0.05 µM versus 0.22 ± 0.03 µM; P = 0.006), demonstrating that modulation of i2 levels meaningfully impacts drug bioavailability and cellular response. We also observed reduced production of reactive oxygen species by 30% (P < 0.05), and an enhanced expression (>1.2-fold; P < 0.05) of several proteins, such as hemoglobin α genes and superoxide dismutase 1, that have network functions associated with antioxidant properties. Interaction proteomics analysis of endogenous proteins from the cellular model, mainly in human intestine but also in kidney tissues, further uncovered interactions between i2s (but not i1s) and the antioxidant enzymes catalase and peroxiredoxin 1, which may influence antioxidant potential through sequestration of these novel partners. Our findings demonstrate for the first time dual roles for i2s in the cellular defense system as endogenous regulators of drug response as well as in oxidative stress.
Subject(s)
Alternative Splicing , Glucuronosyltransferase/metabolism , Antineoplastic Agents/pharmacology , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Catalase/genetics , Catalase/metabolism , Cell Line, Tumor , Cell Survival , Colonic Neoplasms , Gene Expression Profiling , Gene Knockdown Techniques , Glucuronosyltransferase/genetics , Humans , Irinotecan , Isoenzymes/genetics , Isoenzymes/metabolism , Oxidative Stress , Peroxiredoxins/genetics , Peroxiredoxins/metabolism , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolismABSTRACT
Alternative splicing (AS) is one of the most significant components of the functional complexity of human UDP-glucuronosyltransferase enzymes (UGTs), particularly for the UGT1A gene, which represents one of the best examples of a drug-metabolizing gene regulated by AS. Shorter UGT1A isoforms [isoform 2 (i2)] are deficient in glucuronic acid transferase activity but function as negative regulators of enzyme activity through protein-protein interaction. Their abundance, relative to active UGT1A enzymes, is expected to be a determinant of the global transferase activity of cells and tissues. Here we tested whether i2-mediated inhibition increases with greater abundance of the i2 protein relative to the isoform 1 (i1) enzyme, using the extrahepatic UGT1A7 as a model and a series of 23 human embryonic kidney 293 clonal cell lines expressing variable contents of i1 and i2 proteins. Upon normalization for i1, a significant reduction of 7-ethyl-10-hydroxycamptothecin glucuronide formation was observed for i1+i2 clones (mean of 53%) compared with the reference i1 cell line. In these clones, the i2 protein content varied greatly (38-263% relative to i1) and revealed two groups: 17 clones with i2 < i1 (60% ± 3%) and 6 clones with i2 ≥ i1 (153% ± 24%). The inhibition induced by i2 was more substantial for clones displaying i2 ≥ i1 (74.5%; P = 0.001) compared with those with i2 < i1 (45.5%). Coimmunoprecipitation supports a more substantial i1-i2 complex formation when i2 exceeds i1. We conclude that the relative abundance of regulatory i2 proteins has the potential to drastically alter the local drug metabolism in the cells, particularly when i2 surpasses the protein content of i1.
Subject(s)
Alternative Splicing , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Camptothecin/analogs & derivatives , Camptothecin/metabolism , Glucuronides/metabolism , Glucuronosyltransferase/analysis , HEK293 Cells , Humans , Isoenzymes/analysis , Isoenzymes/genetics , Isoenzymes/metabolismABSTRACT
The oligomerization of UGTs [UDP (uridine diphosphate)-glucuronosyltransferases] modulates their enzyme activities. Recent findings also indicate that glucuronidation is negatively regulated by the formation of inactive oligomeric complexes between UGT1A enzymes [i1 (isoform 1)] and an enzymatically inactive alternatively spliced i2 (isoform 2). In the present paper, we assessed whether deletion of the UGT-interacting domains previously reported to be critical for enzyme function might be involved in i1-i2 interactions. The bilirubin-conjugating UGT1A1 was used as a prototype. We also explored whether intermolecular disulfide bonds are involved in i1-i2 interactions and the potential role of selected cysteine residues. Co-immunoprecipitation assays showed that UGT1A1 lacking the SP (signal peptide) alone or also lacking the transmembrane domain (absent from i2) did not self-interact, but still interacted with i2. The deletion of other N- or C-terminal domains did not compromise i1-i2 complex formation. Under non-reducing conditions, we also observed formation of HMWCs (high-molecular-mass complexes) for cells overexpressing i1 and i2. The presence of UGTs in these complexes was confirmed by MS. Mutation of individual cysteine residues throughout UGT1A1 did not compromise i1-i1 or i1-i2 complex formation. These findings are compatible with the hypothesis that the interaction between i1 and i2 proteins (either transient or stable) involves binding of more than one domain that probably differs from those involved in i1-i1 interactions.
Subject(s)
Alternative Splicing , Glucuronosyltransferase/chemistry , Glucuronosyltransferase/genetics , Bilirubin/metabolism , Glucuronosyltransferase/metabolism , HEK293 Cells , Humans , Immunoprecipitation , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
PURPOSE: Prostate cancer is a heterogeneous genetic disease, and molecular methods for predicting prognosis in patients with aggressive form of the disease are urgently needed to better personalize treatment approaches. The objective was to identify host genetic variations in candidate steroidogenic genes affecting hormone levels and prostate cancer progression. EXPERIMENTAL DESIGN: The study examined two independent cohorts composed of 526 Caucasian men with organ-confined prostate cancer and 601 Taiwanese men on androgen-deprivation therapy. Caucasians were genotyped for 109 haplotype-tagging single-nucleotide polymorphisms (SNP) in CYP17A1, ESR1, CYP19A1, and HSD3B1, and their prognostic significance on disease progression was assessed using Kaplan-Meier survival curves and Cox regression models. Positive findings, including previously identified SRD5A1, SRD5A2, HSD17B2, HSD17B3, and HSD17B12 polymorphisms, were then explored in Taiwanese men (n = 32 SNPs). The influence of positive markers on the circulating hormonal levels was then appraised in Caucasians using specific and sensitive mass spectrometry-based methods. RESULTS: After adjusting for known risk factors, variants of CYP17A1 (rs6162), HSD17B2 (rs4243229 and rs7201637), and ESR1 (rs1062577) were associated with progressive disease in both cohorts. Indeed, the presence of these variations was significantly associated with progression in Caucasians (HR, 2.29-4.10; P = 0.0014-2 × 10(-7)) and survival in Taiwanese patients [HR = 3.74; 95% confidence interval (CI): 1.71-8.19, P = 0.009]. Remarkably, the CYP17A1 rs6162 polymorphism was linked to plasma dehydroepiandrosterone-sulfate (DHEA-S) levels (P = 0.03), HSD17B2 rs7201637 with levels of dihydrotestosterone (P = 0.03), and ESR1 rs1062577 with levels of estrone-S and androsterone-glucuronide (P ≤ 0.05). CONCLUSION: This study identifies, in different ethnic groups and at different disease stages, CYP17A1, HSD17B2, and ESR1 as attractive prognostic molecular markers of prostate cancer progression.
Subject(s)
Biomarkers, Tumor , Prostatic Neoplasms/metabolism , Signal Transduction , Steroids/metabolism , Adult , Aged , Aged, 80 and over , Alleles , Asian People/genetics , Disease Progression , Genotype , Gonadal Steroid Hormones/blood , Gonadal Steroid Hormones/metabolism , Humans , Male , Middle Aged , Neoplasm Grading , Neoplasm Staging , Polymorphism, Single Nucleotide , Prostatic Neoplasms/genetics , Prostatic Neoplasms/mortality , Prostatic Neoplasms/pathology , Steroids/blood , White People/geneticsABSTRACT
Glucuronidation by UDP-glucuronyltransferase (UGT) enzymes is the prevailing conjugative pathway for the metabolism of both xenobiotics and endogenous compounds. Alterations in this pathway, such as those generated by common genetic polymorphisms, have been shown to significantly impact on the health of individuals, influencing cancer susceptibility, responsiveness to drugs and drug-induced toxicity. Alternative usage of terminal exons leads to UGT1A-derived splice variants, namely the classical and enzymatically active isoforms 1 (i1) and the novel enzymatically inactive isoforms 2 (i2). In vitro functional data from heterologous expression and RNA interference experiments indicate that these i2 isoforms act as negative modulators of glucuronidation, likely by forming inactive complexes with active isoform 1. We used specific antibodies against either active i1 or inactive i2 proteins to examine their distribution in major drug-metabolizing tissues. Data revealed that UGT1A_i1 and inactive UGT1A_i2 are co-produced in the same tissue structures, including liver, kidney, stomach, intestine and colon. Examination of the cellular distribution and semi-quantitative level of expression of UGT1As revealed heterogeneous expression of i1 and i2 proteins, with increased expression of i2 in liver tumours and decreased levels of i1 and i2 in colon cancer specimens, compared to normal tissues. These differences in expression may be relevant to human colon and liver cancer tumorigenesis. Our data clearly demonstrate the similar immunolocalization of active and inactive UGT1A isoforms in most UGT1A-expressing cell types of major tissues involved in drug metabolism. These expression patterns are consistent with a dominant-negative function for the i2 encoded by the UGT1A gene.
Subject(s)
Colonic Neoplasms/enzymology , Glucuronosyltransferase/metabolism , Liver Neoplasms/enzymology , Alternative Splicing , Humans , Intestine, Small/enzymology , Isoenzymes/metabolism , Kidney/enzymology , Stomach/enzymologyABSTRACT
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
Subject(s)
Alternative Splicing , Glucuronosyltransferase/antagonists & inhibitors , Glucuronosyltransferase/genetics , Multienzyme Complexes/metabolism , Blotting, Western , Cell Line , Colon/enzymology , Esophagus/enzymology , Humans , Immunoprecipitation , Intestine, Small/enzymology , Liver/enzymology , Protein Isoforms , Protein Multimerization , TransfectionABSTRACT
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.