ABSTRACT
Malignant peripheral nerve sheath tumor is a rare and aggressive disease with poor treatment response, mainly affecting adolescents and young adults. Few molecular biomarkers are used in the management of this cancer type, and although TP53 is one of few recurrently mutated genes in malignant peripheral nerve sheath tumor, the mutation prevalence and the corresponding clinical value of the TP53 network remains unsettled. We present a multi-level molecular study focused on aberrations in the TP53 network in relation to patient outcome in a series of malignant peripheral nerve sheath tumors from 100 patients and 38 neurofibromas, including TP53 sequencing, high-resolution copy number analyses of TP53 and MDM2, and gene expression profiling. Point mutations in TP53 were accompanied by loss of heterozygosity, resulting in complete loss of protein function in 8.2% of the malignant peripheral nerve sheath tumors. Another 5.5% had MDM2 amplification. TP53 mutation and MDM2 amplification were mutually exclusive and patients with either type of aberration in their tumor had a worse prognosis, compared to those without (hazard ratio for 5-year disease-specific survival 3.5, 95% confidence interval 1.78-6.98). Both aberrations had similar consequences on the gene expression level, as analyzed by a TP53-associated gene signature, a property also shared with the copy number aberrations and/or loss of heterozygosity at the TP53 locus, suggesting a common "TP53-mutated phenotype" in as many as 60% of the tumors. This was a poor prognostic phenotype (hazard ratio = 4.1, confidence interval:1.7-9.8), thus revealing a TP53-non-aberrant patient subgroup with a favorable outcome. The frequency of the "TP53-mutated phenotype" warrants explorative studies of stratified treatment strategies in malignant peripheral nerve sheath tumor.
Subject(s)
Nerve Sheath Neoplasms/genetics , Nerve Sheath Neoplasms/pathology , Neurofibrosarcoma/genetics , Neurofibrosarcoma/pathology , Tumor Suppressor Protein p53/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Child , Female , Gene Amplification , Genes, p53/genetics , Humans , Male , Middle Aged , Mutation , Nerve Sheath Neoplasms/mortality , Neurofibrosarcoma/mortality , Prognosis , Proto-Oncogene Proteins c-mdm2/genetics , Young AdultABSTRACT
The connexins constitute a family of integral membrane proteins that form channels between adjacent cells. These channels are assembled in plasma membrane domains known as gap junctions and enable cells to directly exchange ions and small molecules. Intercellular communication via gap junctions plays important roles in regulating cell growth and differentiation and in maintaining tissue homeostasis. This type of cell communication is often impaired during cancer development, and several members of the connexin protein family have been shown to act as tumor suppressors. Emerging evidence suggests that the connexin protein family has important roles in colorectal cancer development. In the normal colonic epithelial tissue, three connexin isoforms, connexin 26 (Cx26), Cx32 and Cx43, have been shown to be expressed at the protein level. Colorectal cancer development is associated with loss of connexin expression or relocalization of connexins from the plasma membrane to intracellular compartments. Downregulation of connexins in colorectal carcinomas at the transcriptional level involves cancer-specific promoter hypermethylation. Recent studies suggest that Cx43 may constrain growth of colon cancer cells by interfering with the Wnt/ß-catenin pathway. There is also increasing evidence that the connexins may have potential as prognostic markers in colorectal cancer. This review discusses the role of connexins in colorectal cancer pathogenesis, as well as their potential as prognostic markers and targets in the prevention and treatment of the disease.
Subject(s)
Biomarkers, Tumor/metabolism , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Connexins/metabolism , Biomarkers, Tumor/genetics , Colorectal Neoplasms/therapy , Connexin 26 , Connexins/genetics , DNA Methylation , Gene Expression Regulation, Neoplastic , Humans , Molecular Targeted Therapy , Wnt Signaling PathwayABSTRACT
SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.
Subject(s)
Connexin 43/metabolism , Gap Junctions/metabolism , Lysine/metabolism , Protein Processing, Post-Translational , Animals , Blotting, Western , Cell Membrane/metabolism , Connexin 43/genetics , HeLa Cells , Humans , Lysine/genetics , Microscopy, Confocal , Mutation , Rats , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , TransfectionABSTRACT
BACKGROUND: Malignant peripheral nerve sheath tumour (MPNST) is an aggressive orphan disease commonly affecting adolescents or young adults. Current knowledge of molecular tumour biology has been insufficient for development of rational treatment strategies. We aimed to discover molecular subtypes of potential clinical relevance. METHODS: Fresh frozen samples of MPNSTs (n = 94) and benign neurofibromas (n = 28) from 115 patients in a European multicentre study were analysed by DNA copy number and/or transcriptomic profiling. Unsupervised transcriptomic subtyping was performed and the subtypes characterized for genomic aberrations, clinicopathological associations and patient survival. FINDINGS: MPNSTs were classified into two transcriptomic subtypes defined primarily by immune signatures and proliferative processes. "Immune active" MPNSTs (44%) had sustained immune signals relative to neurofibromas, were more frequently low-grade (P = 0.01) and had favourable prognostic associations in a multivariable model of disease-specific survival with clinicopathological factors (hazard ratio 0.25, P = 0.003). "Immune deficient" MPNSTs were more aggressive and characterized by proliferative signatures, high genomic complexity, aberrant TP53 and PRC2 loss, as well as high relative expression of several potential actionable targets (EGFR, ERBB2, EZH2, KIF11, PLK1, RRM2). Integrated gene-wise analyses suggested a DNA copy number-basis for proliferative transcriptomic signatures in particular, and the tumour copy number burden further stratified the transcriptomic subtypes according to patient prognosis (P < 0.01). INTERPRETATION: Approximately half of MPNSTs belong to an "immune deficient" transcriptomic subtype associated with an aggressive disease course, PRC2 loss and expression of several potential therapeutic targets, providing a rationale for molecularly-guided intervention trials. FUNDING: Research grants from non-profit organizations, as stated in the Acknowledgements.
Subject(s)
Nerve Sheath Neoplasms , Neurofibroma , Neurofibrosarcoma , Adolescent , Young Adult , Humans , Nerve Sheath Neoplasms/diagnosis , Nerve Sheath Neoplasms/genetics , Nerve Sheath Neoplasms/metabolism , Transcriptome , Neurofibroma/genetics , Neurofibroma/pathology , Genomics , DNAABSTRACT
This article is the first to show that loss of connexin43 (Cx43) expression in colorectal tumors is correlated with significantly shorter relapse-free and overall survival. Cx43 was further found to negatively regulate growth of colon cancer cells, in part by enhancing apoptosis. In addition, Cx43 was found to colocalize with ß-catenin and reduce Wnt signaling. The study represents the first evidence that Cx43 acts as a colorectal cancer tumor suppressor and that loss of Cx43 expression during colorectal cancer development is associated with reduced patient survival. The study has important implications for the assessment of Cx43 as a prognostic marker and target in colorectal cancer prevention and therapy. Gap junctions consist of intercellular channels that permit direct transfer of ions and small molecules between adjacent cells. The gap junction channel protein Cx43 plays important roles in cell growth control and differentiation and is frequently dysregulated in human cancers. However, the functional importance and clinical relevance of Cx43 in cancer development has remained elusive. Here, we show that Cx43 is downregulated or aberrantly localized in colon cancer cell lines and colorectal carcinomas, which is associated with loss of gap junction intercellular communication. The in situ protein expression of Cx43 was analyzed in colorectal tumors in a cohort of 674 patients and related to established clinicopathological variables and survival. A subgroup of the patients had weak or no expression of Cx43 in tumors. Loss of Cx43 expression was significantly correlated with shorter relapse-free and overall survival. Loss of Cx43 further identified a high-risk subgroup among stage I and stage II patients with reduced relapse-free and overall survival. Ectopic expression of Cx43 in the colon cancer cell line HT29 was associated with reduced growth in monolayer and soft agar cultures and in tumor xenografts. Cx43 was found to colocalize with ß-catenin and negatively regulate the Wnt signaling pathway, and expression of Cx43 was associated with increased levels of apoptosis. Altogether, these data indicate that Cx43 is a colorectal cancer tumor suppressor protein that predicts clinical outcome.
Subject(s)
Colorectal Neoplasms/metabolism , Colorectal Neoplasms/mortality , Connexin 43/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Annexin A5/analysis , Apoptosis , Biomarkers, Tumor , Cell Communication , Cell Line, Tumor , Colorectal Neoplasms/pathology , Connexin 43/genetics , Disease-Free Survival , Female , Gap Junctions/metabolism , Gap Junctions/physiology , HT29 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Prognosis , Survival Rate , Transplantation, Heterologous , Tumor Suppressor Proteins/genetics , Wnt Signaling Pathway , beta Catenin/metabolismABSTRACT
BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate the incidence of peritoneal carcinomatosis (PC) in a prospectively recorded series of colon cancer patients from a defined cohort and to compare clinicopathological characteristics, survival, and TP53 mutation status in primary tumors from patients with and without PC. METHODS: Clinical data from all colon cancer patients admitted in 1993-2006 were registered prospectively (n = 1,124). In a subset of PC patients, DNA was retrieved from tumor tissue and TP53 mutations analyzed and compared to the mutation status in a historical series. RESULTS: In the prospective series 10% of female and 7% of male patients had PC (P = 0.05). The PC patients were younger than those without PC (median 4 years, P = 0.002). The incidence of PC was 10.3% and 6.2% (P = 0.03) in patients with primary tumors in the right and left colon, respectively. TP53 was mutated in 57% of the PC patients as compared to 41% in the series of patients without PC (P = 0.05). CONCLUSIONS: The incidence of PC was higher in right-sided colon cancer and among women. PC patients were younger than non-PC patients, and PC was independently associated with TP53 mutation in the primary tumor.
Subject(s)
Carcinoma/epidemiology , Carcinoma/genetics , Colonic Neoplasms/pathology , Genes, p53/genetics , Mutation , Peritoneal Neoplasms/epidemiology , Peritoneal Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Carcinoma/secondary , Case-Control Studies , Colonic Neoplasms/epidemiology , DNA Mutational Analysis , DNA, Neoplasm/analysis , Female , Humans , Incidence , Male , Middle Aged , Multivariate Analysis , Norway/epidemiology , Peritoneal Neoplasms/secondary , Prospective Studies , Sex DistributionSubject(s)
Antibodies/chemistry , Artifacts , Indicators and Reagents/chemistry , Nuclear Proteins/analysis , Phosphoprotein Phosphatases/analysis , beta Catenin/analysis , Antibodies/immunology , Caco-2 Cells , Cross Reactions/immunology , False Positive Reactions , Gene Expression , Humans , Immunohistochemistry , Indicators and Reagents/standards , Mass Spectrometry , Nuclear Proteins/genetics , Phosphoprotein Phosphatases/genetics , Proto-Oncogene Mas , beta Catenin/geneticsABSTRACT
BACKGROUND: There is a need for improved selection of patients for adjuvant chemotherapy after resection of non-metastatic colorectal cancer (CRC). Regulator of chromosome condensation 2 (RCC2) is a potential prognostic biomarker. We report on the establishment of a robust protocol for RCC2 expression analysis and prognostic tumour biomarker evaluation in patients who did and did not receive adjuvant chemotherapy. MATERIALS AND METHODS: RCC2 was analysed in 2916 primary CRCs from the QUASAR2 randomised trial and two single-hospital Norwegian series. A new protocol using fluorescent antibody staining and digital image analysis was optimised. Biomarker value for 5-year relapse-free survival was analysed in relation to tumour stage, adjuvant chemotherapy and the molecular markers microsatellite instability, KRAS/BRAFV600E/TP53 mutations and CDX2 expression. RESULTS: Low RCC2 expression was scored in 41% of 2696 evaluable samples. Among patients with stage I-III CRC who had not received adjuvant chemotherapy, low RCC2 expression was an independent marker of inferior 5-year relapse-free survival in multivariable Cox models including clinicopathological factors and molecular markers (HR 1.45, 95% CI 1.09 to 1.94, p=0.012, N=521). RCC2 was not prognostic in patients who had received adjuvant chemotherapy, neither in QUASAR2 nor the pooled Norwegian series. The interaction between RCC2 and adjuvant chemotherapy for prediction of patient outcome was significant in stage III, and strongest among patients with microsatellite stable tumours (pinteraction=0.028). CONCLUSIONS: Low expression of RCC2 is a biomarker for poor prognosis in patients with stage I-III CRC and seems to be a predictive biomarker for effect of adjuvant chemotherapy.
Subject(s)
Colorectal Neoplasms , Neoplasm Recurrence, Local , Chemotherapy, Adjuvant , Chromosomal Proteins, Non-Histone/therapeutic use , Chromosomes , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Guanine Nucleotide Exchange Factors/therapeutic use , Humans , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Neoplasm StagingABSTRACT
The purpose of this study was to identify new prognostic biomarkers with clinical impact in malignant peripheral nerve sheath tumor (MPNST), a highly aggressive malignancy for which no consensus therapy exists besides surgery. We have used tissue microarrays (TMAs) to assess in situ expression of 14 cell-cycle-regulating proteins in 64 well-characterized MPNST patients: 36 sporadic and 28 with neurofibromatosis type 1 (NF1). We developed a new software application for evaluation and logistics of the TMA images and performed a literature survey of cell cycle proteins in MPNST. For NF1-associated patients, there was a clear association between nuclear expression of p53 and poor survival (p = 0.004). Among the other proteins analyzed, we also found significant associations between survival and clinical variables, but none were as strong as that for p53. For the total series of MPNSTs, p53 was shown to be an independent predictor of survival, and patients without remission, with tumor size larger than 8 cm, and with positive p53 expression had a 60 times greater risk of dying within the first 5 years compared with the remaining patients (p = 0.000002). This is the most comprehensive study of in situ protein expression in MPNST so far, and expressed p53 was found to be a strong surrogate marker for outcome. Patients in complete remission with a primary p53-positive MPNST diagnosis may be considered in a high-risk subgroup and candidates for adjuvant treatment.
Subject(s)
Biomarkers, Tumor/analysis , Nerve Sheath Neoplasms/mortality , Nerve Sheath Neoplasms/pathology , Tumor Suppressor Protein p53/biosynthesis , Adult , Blotting, Western , Comparative Genomic Hybridization , Disease-Free Survival , Female , Gene Expression , Humans , Image Processing, Computer-Assisted/methods , Immunohistochemistry , Kaplan-Meier Estimate , Male , Middle Aged , Nerve Sheath Neoplasms/metabolism , Software , Tissue Array AnalysisABSTRACT
BACKGROUND: Tumor-derived aberrantly methylated DNA might serve as diagnostic biomarkers for cancer, but so far, few such markers have been identified. The aim of the present study was to investigate the potential of the MAL (T-cell differentiation protein) gene as an early epigenetic diagnostic marker for colorectal tumors. METHODS: Using methylation-specific polymerase chain reaction (MSP) the promoter methylation status of MAL was analyzed in 218 samples, including normal mucosa (n = 44), colorectal adenomas (n = 63), carcinomas (n = 65), and various cancer cell lines (n = 46). Direct bisulphite sequencing was performed to confirm the MSP results. MAL gene expression was investigated with real time quantitative analyses before and after epigenetic drug treatment. Immunohistochemical analysis of MAL was done using normal colon mucosa samples (n = 5) and a tissue microarray with 292 colorectal tumors. RESULTS: Bisulphite sequencing revealed that the methylation was unequally distributed within the MAL promoter and by MSP analysis a region close to the transcription start point was shown to be hypermethylated in the majority of colorectal carcinomas (49/61, 80%) as well as in adenomas (45/63, 71%). In contrast, only a minority of the normal mucosa samples displayed hypermethylation (1/23, 4%). The hypermethylation of MAL was significantly associated with reduced or lost gene expression in in vitro models. Furthermore, removal of the methylation re-induced gene expression in colon cancer cell lines. Finally, MAL protein was expressed in epithelial cells of normal colon mucosa, but not in the malignant cells of the same type. CONCLUSION: Promoter hypermethylation of MAL was present in the vast majority of benign and malignant colorectal tumors, and only rarely in normal mucosa, which makes it suitable as a diagnostic marker for early colorectal tumorigenesis.
Subject(s)
Biomarkers, Tumor/metabolism , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , DNA Methylation , Membrane Transport Proteins/genetics , Myelin Proteins/genetics , Proteolipids/genetics , Colorectal Neoplasms/metabolism , Humans , Myelin and Lymphocyte-Associated Proteolipid Proteins , Promoter Regions, GeneticABSTRACT
We aimed to refine the value of CDX2 as an independent prognostic and predictive biomarker in colorectal cancer (CRC) according to disease stage and chemotherapy sensitivity in preclinical models. CDX2 expression was evaluated in 1045 stage I-IV primary CRCs by gene expression (n = 403) or immunohistochemistry (n = 642) and in relation to 5-year relapse-free survival (RFS), overall survival (OS), and chemotherapy. Pharmacogenomic associations between CDX2 expression and 69 chemotherapeutics were assessed by drug screening of 35 CRC cell lines. CDX2 expression was lost in 11.6% of cases and showed independent poor prognostic value in multivariable models. For individual stages, CDX2 was prognostic only in stage IV, independent of chemotherapy. Among stage I-III patients not treated in an adjuvant setting, CDX2 loss was associated with a particularly poor survival in the BRAF-mutated subgroup, but prognostic value was independent of microsatellite instability status and the consensus molecular subtypes. In stage III, the 5-year RFS rate was higher among patients with loss of CDX2 who received adjuvant chemotherapy than among patients who did not. The CDX2-negative cell lines were significantly more sensitive to chemotherapeutics than CDX2-positive cells, and the multidrug resistance genes MDR1 and CFTR were significantly downregulated both in CDX2-negative cells and in patient tumors. Loss of CDX2 in CRC is an adverse prognostic biomarker only in stage IV disease and appears to be associated with benefit from adjuvant chemotherapy in stage III. Early-stage patients not qualifying for chemotherapy might be reconsidered for such treatment if their tumor has loss of CDX2 and mutated BRAF.
Subject(s)
Biomarkers, Tumor/genetics , CDX2 Transcription Factor/genetics , Colorectal Neoplasms/drug therapy , Pharmacogenomic Testing , Chemotherapy, Adjuvant , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Disease-Free Survival , Female , Gene Expression , Hospitals, University , Humans , Kaplan-Meier Estimate , Male , Microsatellite Instability , Multivariate Analysis , Mutation , Neoplasm Staging , Norway , Prognosis , Proportional Hazards Models , Proto-Oncogene Proteins B-raf/genetics , Registries , Retrospective Studies , Survival AnalysisABSTRACT
Patients with malignant peripheral nerve sheath tumor (MPNST), a rare soft tissue cancer associated with loss of the tumor suppressor neurofibromin (NF1), have poor prognosis and typically respond poorly to adjuvant therapy. We evaluated the effect of 299 clinical and investigational compounds on seven MPNST cell lines, two primary cultures of human Schwann cells, and five normal bone marrow aspirates, to identify potent drugs for MPNST treatment with few side effects. Top hits included Polo-like kinase 1 (PLK1) inhibitors (volasertib and BI2536) and the fluoronucleoside gemcitabine, which were validated in orthogonal assays measuring viability, cytotoxicity, and apoptosis. DNA copy number, gene expression, and protein expression were determined for the cell lines to assess pharmacogenomic relationships. MPNST cells were more sensitive to BI2536 and gemcitabine compared to a reference set of 94 cancer cell lines. PLK1, RRM1, and RRM2 mRNA levels were increased in MPNST compared to benign neurofibroma tissue, and the protein level of PLK1 was increased in the MPNST cell lines compared to normal Schwann cells, indicating an increased dependence on these drug targets in malignant cells. Furthermore, we observed an association between increased mRNA expression of PLK1, RRM1, and RRM2 in patient samples and worse disease outcome, suggesting a selective benefit from inhibition of these genes in the most aggressive tumors.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Deoxycytidine/analogs & derivatives , Drug Resistance, Neoplasm , Nerve Sheath Neoplasms/drug therapy , Nerve Sheath Neoplasms/enzymology , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Apoptosis/drug effects , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Clinical Trials as Topic , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Nerve Sheath Neoplasms/genetics , Nerve Sheath Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Reproducibility of Results , Ribonucleoside Diphosphate Reductase/genetics , Ribonucleoside Diphosphate Reductase/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Gemcitabine , Polo-Like Kinase 1ABSTRACT
Ribonucleotide reductase (RNR) is the enzyme responsible for the conversion of ribonucleotides to 2'-deoxyribonucleotides and thereby provides the precursors needed for both synthesis and repair of DNA. In the recent years, many new crystal structures have been obtained of the protein subunits of all three classes of RNR. This review will focus upon recent structural and spectroscopic studies, which have offered deeper insight to the mechanistic properties as well as evolutionary relationship and diversity among the different classes of RNR. Although the three different classes of RNR enzymes depend on different metal cofactors for the catalytic activity, all three classes have a conserved cysteine residue at the active site located on the tip of a protein loop in the centre of an alpha/beta-barrel structural motif. This cysteine residue is believed to be converted into a thiyl radical that initiates the substrate turnover in all three classes of RNR. The functional and structural similarities suggest that the present-day RNRs have all evolved from a common ancestral reductase. Nevertheless, the different cofactors found in the three classes of RNR make the RNR proteins into interesting model systems for quite diverse protein families, such as diiron-oxygen proteins, cobalamin-dependent proteins, and SAM-dependent iron-sulfur proteins. There are also significant variations within each of the three classes of RNR. With new structures available of the R2 protein of class I RNR, we have made a comparison of the diiron centres in R2 from mouse and Escherichia coli. The R2 protein shows dynamic carboxylate, radical, and water shifts in different redox forms, and new radical forms are different from non-radical forms. In mouse R2, the binding of iron(II) or cobalt(II) to the four metal sites shows high cooperativity. A unique situation is found in RNR from baker's yeast, which is made up of heterodimers, in contrast to homodimers, which is the normal case for class I RNR. Since the reduction of ribonucleotides is the rate-limiting step of DNA synthesis, RNR is an important target for cell growth control, and the recent finding of a p53-induced isoform of the R2 protein in mammalian cells has increased the interest for the role of RNR during the different phases of the cell cycle.
Subject(s)
Enzyme Inhibitors/pharmacology , Ribonucleotide Reductases/chemistry , Ribonucleotide Reductases/physiology , Allosteric Regulation , Animals , Binding Sites , Catalysis , Drug Design , Humans , Iron/metabolism , Oxidation-Reduction , Protein ConformationABSTRACT
Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH(4))-dependent enzyme that catalyzes the hydroxylation of l-Phe to l-Tyr. The non-heme iron in the enzyme (Fe(III) as isolated) is 6-coordinated to a 2-His-1-carboxylate motif and three water molecules (wat1, wat2 and wat3). Tyr325 is at the second coordination sphere, hydrogen-bonded to water (wat1). We prepared and expressed mutants with Leu, Ala, Ser and Phe at this position. Only Y325L and the conservative mutation Y325F resulted in stable enzymes, but the mutant Y325F has been found to be post-translationally hydroxylated and to revert back to wild-type PAH [S.D. Kinzie, M. Thevis, K. Ngo, J. Whitelegge, J.A. Loo, M.M. Abu-Omar, J. Am. Chem. Soc. 125 (2003) 4710-4711], being inadequate to investigate the early inferred functional role of Tyr325. On the other hand, compared to wild-type PAH, Y325L shows reduced specific activity, decreased coupling efficiency and decreased iron content. The mutant also reveals a very high affinity for l-Phe and BH(4) and does not manifest positive cooperativity for the substrate. All together, our results support that the mutation Y325L causes the removal or increased delocalization of the iron-ligated wat1 and, in turn, a less tight binding of the metal. Tyr325 thus appears to have an important role ensuring stoichiometric binding of iron, correct geometry of the complexes with substrate and cofactor and, consequently, a right coupling efficiency of the PAH reaction. In addition, the residue appears to be important for the correct cooperative regulation by l-Phe.
Subject(s)
Iron/metabolism , Phenylalanine Hydroxylase/chemistry , Phenylalanine Hydroxylase/metabolism , Base Sequence , Catalytic Domain/genetics , DNA, Recombinant/genetics , Electron Spin Resonance Spectroscopy , Humans , Hydroxylation , In Vitro Techniques , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Phenylalanine Hydroxylase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Tyrosine/chemistryABSTRACT
Three microperoxidases--hemin-6(7)-gly-gly-his methyl ester (HGGH), mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH)--have been prepared as models for heme-containing peroxidases by condensation of glycyl-glycyl-L-histidine methyl ester with the propionic side chains of hemin, mesohemin and deuterohemin, respectively. The three microperoxidases differ in two substituents, R, of the protoporphyrin IX framework (HGGH: R=vinyl, MGGH: R=ethyl, DGGH: R=H). X-band and high field EPR spectra show that the microperoxidases exhibit spectroscopic properties similar to those of metmyoglobin, i.e. a high spin ferric S=5/2 signal at g(perpendicular)=6 and g parallel)=2 and an estimated D value of 7.5+/-1cm(-1). The catalytic activities of the microperoxidases towards K4[Fe(CN)6], L-tyrosine methyl ester and 2,2'-azino(bis(3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS) have been investigated. It was found that all three microperoxidases exhibit peroxidase activity and that the reactions follow the generally accepted peroxidase reaction scheme [Biochem. J. 145 (1975) 93-103] with the exception that the initial formation of a Compound I analogue is the rate-limiting step for the whole process. The general activity trend was found to be MGGH approximately DGGH>HGGH. For each microperoxidase, DFT calculations (B3LYP) were made on the reactions of compounds 0, I and II with H+, e- and H+ + e-, respectively, in order to probe the possible relationship between the nature of the 2- and 4-substituents of the hemin and the observed reactivity. The computational modeling indicates that the relative energy differences are very small; solvation and electrostatic effects may be factors that decide the relative activities of the microperoxidases.
Subject(s)
Heme/metabolism , Hemin/analogs & derivatives , Hemin/metabolism , Mesoporphyrins/metabolism , Peroxidases/metabolism , Tyrosine/analogs & derivatives , Benzothiazoles , Computational Biology , Dipeptides/chemistry , Electrochemistry , Hydrogen Peroxide/chemistry , Kinetics , Magnetic Resonance Spectroscopy , Models, Biological , Oligopeptides/chemical synthesis , Sulfonic Acids/chemistry , Tyrosine/chemistryABSTRACT
BACKGROUND: Malignant peripheral nerve sheath tumor (MPNST) is a rare and highly aggressive disease with no evidence of effect from adjuvant therapy. It is further associated with the hereditary syndrome neurofibromatosis type 1 (NF1). Silencing of the tumor suppressor gene RASSF1A through DNA promoter hypermethylation is known to be involved in cancer development, but its impact in MPNSTs remains unsettled. METHODS: The RASSF1A promoter was analyzed by methylation-specific PCR in 113 specimens, including 44 NF1-associated MPNSTs, 47 sporadic MPNSTs, 21 benign neurofibromas, and 1 nonneoplastic nerve sheath control. RESULTS: RASSF1A methylation was found only in the malignant samples (60%) and identified a subgroup among patients with NF1-associated MPNST with a poor prognosis. These patients had a mean 5-year disease-specific survival of 27.3 months (95% CI: 17.2-37.4) versus 47.4 months (95% CI: 37.5-57.2) for NF1 patients with unmethylated promoters, P = 0.014. In multivariate Cox regression analysis, methylated RASSF1A remained an adverse prognostic factor independent of clinical risk factors, P = .013 (hazard ratio: 5.2; 95% CI: 1.4-19.4). CONCLUSION: A considerable number of MPNST samples display hypermethylation of the RASSF1A gene promoter, and for these tumors, this is the first molecular marker that if validated can characterize a subgroup of patients with inferior prognosis, restricted to individuals with NF1.
Subject(s)
DNA Methylation , Neurilemmoma/genetics , Neurofibromatosis 1/genetics , Tumor Suppressor Proteins/genetics , Adolescent , Adult , Aged , Child , Female , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Neurilemmoma/complications , Neurofibromatosis 1/complications , Prognosis , Promoter Regions, Genetic , Young AdultABSTRACT
No consensus treatment regime exists beyond surgery for malignant peripheral nerve sheath tumours (MPNST), and the purpose of the present study was to find new approaches to stratify patients with good and poor prognosis and to better guide therapeutic intervention for this aggressive soft tissue cancer. From a total of 67 MPNSTs from Scandinavian patients with and without neurofibromatosis type 1, 30 MPNSTs were investigated by genome-wide RNA expression profiling and 63 MPNSTs by immunohistochemical (IHC) analysis, and selected genes were submitted to analyses of disease-specific survival. The potential drug target genes survivin (BIRC5), thymidine kinase 1 (TK1), and topoisomerase 2-alpha (TOP2A), all encoded on chromosome arm 17q, were up-regulated in MPNST as compared to benign neurofibromas. Each of them was found to be independent prognostic markers on the gene expression level, as well as on the protein level. A prognostic profile was identified by combining the nuclear expression scores of the three proteins. For patients with completely resected tumours only 15% in the high risk group were alive after two years, as compared to 78% in the low risk group. In conclusion, we found a novel protein expression profile which identifies MPNST patients with inferior prognosis even after assumed curative surgery. The tested proteins are drug targets; therefore the expression profile may provide predictive information guiding the design of future clinical trials. Importantly, as the effect is seen on the protein level using IHC, the biomarker panel can be readily implemented in routine clinical testing.
Subject(s)
Antigens, Neoplasm/biosynthesis , DNA Topoisomerases, Type II/biosynthesis , DNA-Binding Proteins/biosynthesis , Gene Expression Regulation, Neoplastic , Inhibitor of Apoptosis Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Neurilemmoma , Thymidine Kinase/biosynthesis , Adolescent , Adult , Aged , Aged, 80 and over , Disease-Free Survival , Female , Genome-Wide Association Study , Humans , Male , Middle Aged , Neurilemmoma/metabolism , Neurilemmoma/mortality , Neurilemmoma/pathology , Neurilemmoma/surgery , Poly-ADP-Ribose Binding Proteins , Retrospective Studies , Survival Rate , SurvivinABSTRACT
PURPOSE: Colorectal cancer has high incidence and mortality worldwide. Patients with microsatellite instable (MSI) tumors have significantly better prognosis than patients with microsatellite stable (MSS) tumors. Considerable variation in disease outcome remains a challenge within each subgroup, and our purpose was to identify biomarkers that improve prediction of colorectal cancer prognosis. EXPERIMENTAL DESIGN: Mutation analyses of 42 MSI target genes were performed in two independent MSI tumor series (n = 209). Markers that were significantly associated with prognosis in the test series were assessed in the validation series, followed by functional and genetic explorations. The clinical potential was further investigated by immunohistochemistry in a population-based colorectal cancer series (n = 903). RESULTS: We identified the cell-cycle gene regulator of chromosome condensation 2 (RCC2) as a cancer biomarker. We found a mutation in the 5' UTR region of RCC2 that in univariate and multivariate analyses was significantly associated with improved outcome in the MSI group. This mutation caused reduction of protein expression in dual luciferase gene reporter assays. siRNA knockdown in MSI colon cancer cells (HCT15) caused reduced cell proliferation, cell-cycle arrest, and increased apoptosis. Massive parallel sequencing revealed few RCC2 mutations in MSS tumors. However, weak RCC2 protein expression was significantly associated with poor prognosis, independent of clinical high-risk parameters, and stratifies clinically important patient subgroups with MSS tumors, including elderly patients (>75 years), stage II patients, and those with rectal cancer. CONCLUSIONS: Impaired RCC2 affects functional and clinical endpoints of colorectal cancer. High-risk patients with either MSI or MSS tumors can be identified with cost-effective routine RCC2 assays.
Subject(s)
Biomarkers, Tumor/genetics , Chromosomal Proteins, Non-Histone/genetics , Colorectal Neoplasms/genetics , Guanine Nucleotide Exchange Factors/genetics , Microsatellite Instability , Age Factors , Aged , Aged, 80 and over , Biomarkers, Tumor/biosynthesis , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/biosynthesis , Chromosomes/genetics , Colorectal Neoplasms/pathology , DNA Mutational Analysis , Disease-Free Survival , Female , Guanine Nucleotide Exchange Factors/biosynthesis , Humans , Male , Middle Aged , Neoplasm Staging , PrognosisABSTRACT
The identification of radicals trapped in irradiated drugs can be very intricate. A multi-frequency electron paramagnetic resonance (EPR) study is proposed to resolve this problem. The Q-band (ca. 34 GHz) comparison with X-band (ca. 9 GHz) did not show significant differences for the four beta-blockers studied (atenolol, esmolol, nadolol and propranolol). The use of a higher frequency (285 GHz) was required. It enabled us to determine the g-tensor values of the radicals present in atenolol and esmolol, respectively, g1 = 2.0086, g2 = 2.0059 and g3 = 2.0021 and g1 = 2.0066, g2 = 2.0044 and g3 = 2.0021. The latter was assigned as a phenoxyl radical, which can not be the case for the former. Therefore, radicals produced in esmolol may result from a more complex mechanism than the abstraction followed by the diffusion of an H atom inside the solid. In addition, two molecules as similar as atenolol and esmolol hydrochloride do not contain the same radicals after irradiation. These two conclusions drawn from the EPR results on beta-blockers show clearly the importance of continuing the investigations on radiolytic mechanisms in solid-state drugs.
Subject(s)
Adrenergic beta-Antagonists/radiation effects , Electron Spin Resonance Spectroscopy/methods , Free Radicals/analysis , Atenolol/radiation effects , Electron Spin Resonance Spectroscopy/instrumentation , Nadolol/radiation effects , Propanolamines/radiation effects , Propranolol/radiation effects , TemperatureABSTRACT
Robust biomarkers that can precisely stratify patients according to treatment needs are in great demand. The literature is inconclusive for most reported prognostic markers for colorectal cancer (CRC). Hence, adequately reported studies in large representative series are necessary to determine their clinical potential. We investigated the prognostic value of three Wnt signaling-associated proteins, ß-catenin, E-cadherin, and SOX9, in a population-representative single-hospital series of 1290 Norwegian CRC patients by performing immunohistochemical analyses of each marker using the tissue microarray technology. Loss of membranous or cytosolic ß-catenin and loss of cytosolic E-cadherin protein expression were significantly associated with reduced 5-year survival in 903 patients who underwent major resection (722 evaluable tissue cores) independently of standard clinicopathological high-risk parameters. Pre-specified subgroup analyses demonstrated particular effect for stage IV patients for ß-catenin membrane staining (P = 0.018; formal interaction test P = 0.025). Among those who underwent complete resection (714 patients, 568 evaluable), 5-year time-to-recurrence analyses were performed, and stage II patients with loss of cytosolic E-cadherin were identified as an independent high-risk subgroup (P = 0.020, formal interaction test was not significant). Nuclear ß-catenin and SOX9 protein, regardless of intracellular location, were not associated with prognosis. In conclusion, the protein expression level of membranous or cytosolic ß-catenin and E-cadherin predicts CRC patient subgroups with inferior prognosis.