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1.
Mol Carcinog ; 59(5): 503-511, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32133692

RESUMO

Lung adenocarcinoma (LUAD), as a form of non-small cell lung cancer (NSCLC), is the most frequently diagnosed lung cancer worldwide. To date, a few biomarkers have been reported to provide valuable information in guiding LUAD treatment. The aim of our study was to explore the functional role of pyrroline-5-carboxylate reductase 1 (PYCR1) in LUAD. Based on Oncomine database, we found that PYCR1 was highly expressed in LUAD tissues. We also confirmed an abnormal increase of PYCR1 expression in LUAD cell lines and patients' tissues. Through Kaplan-Meier plotter database, we further studied the prognostic values of PYCR1. The outcomes indicated that overexpressed PYCR1 associated with poor prognosis among LUAD patients. To further study the function of PYCR1 in LUAD, cell counting kit-8, colony-forming, scratch wound healing, and Transwell assays were conducted. The results suggested that knockdown of PYCR1 curbed cell proliferation, migration, and invasion in LUAD cell lines. Subsequently, we identified 50 top genes positively and negatively correlated with PYCR1 in LUAD, and conducted biological pathway enrichment analysis of these genes. Among those enriched pathways, we selected JAK/STAT signaling pathway for further analysis. The results of Western blot assays revealed that PYCR1 knockdown significantly increased the expression of Bcl-2 and c-Myc, and the phosphorylation level of JAK2 and STAT3. Taken together, this study unearthed that PYCR1 knockdown could inhibit tumor growth and affect the JAK/STAT signaling pathway in LUAD. This study may contribute to a better understanding of PYCR1 in LUAD and provide a potential biomarker for cancer prognosis.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Proliferação de Células , Janus Quinase 2/metabolismo , Pirrolina Carboxilato Redutases/metabolismo , Fator de Transcrição STAT3/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/secundário , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Janus Quinase 2/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Pirrolina Carboxilato Redutases/genética , Fator de Transcrição STAT3/genética , Taxa de Sobrevida , Células Tumorais Cultivadas
2.
Proc Natl Acad Sci U S A ; 117(14): 8083-8093, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213586

RESUMO

Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.


Assuntos
Transformação Celular Neoplásica/patologia , Herpesvirus Humano 8/metabolismo , Prolina/metabolismo , Pirrolina Carboxilato Redutases/metabolismo , Sarcoma de Kaposi/patologia , Proteínas Virais/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Metabolômica , Camundongos , Prolina Oxidase/metabolismo , Sarcoma de Kaposi/virologia , Esferoides Celulares , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Mol Carcinog ; 58(11): 2091-2103, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31435991

RESUMO

Glutamine dependence is a unique metabolic defect seen in cutaneous melanoma (CM), directly influencing the treatment and prognosis. Here, we investigated the associations between 6025 common single-nucleotide polymorphisms (SNPs) in 77 glutamine metabolic pathway genes with CM-specific survival (CMSS) using genotyping datasets from two published genome-wide association studies (GWASs). In the single-locus analysis, 76 SNPs were found to be significantly associated with CMSS (P < .050, false-positive report probability < 0.2 and Bayesian false discovery probability < 0.8) in the discovery dataset, of which seven SNPs were replicated in the validation dataset and three SNPs (HAL rs17676826T > C, LGSN rs12663017T > A, and NOXRED1 rs8012548A > G) independently predicted CMSS, with an effect-allele attributed adjusted hazards ratio of 1.52 (95% confidence interval = 1.19-1.93) and P < .001, 0.68 (0.54-0.87) and P = .002 and 0.62 (0.46-0.83) and P = .002, respectively. The model including the number of unfavorable genotypes (NUGs) of these three SNPs and covariates improved the five-year CMSS prediction (P = .012) than the one with other covariates only. Further expression quantitative trait loci (eQTL) analysis found that the LGSN rs12663017 A allele was significantly associated with increased messenger RNA (mRNA) expression levels (P = 8.89 × 10 -11 ) in lymphoblastoid cell lines of the 1000 Genomes Project database. In the analysis of the genotype tissue expression (GTEx) project datasets, HAL rs17676826 C and NOXRED1 rs8012548 G alleles were significantly associated with their mRNA expression levels in sun-exposed skin of the lower leg (P = 6.62 × 10-6 and 1.37 × 10-7 , respectively) and in sun-not-exposed suprapubic skin (P < .001 and 1.43 × 10-8 , respectively). Taken together, these genetic variants of glutamine-metabolic pathway genes may be promising predictors of survival in patients with CM.


Assuntos
Glutamina/genética , Histidina Amônia-Liase/genética , Melanoma/genética , Pirrolina Carboxilato Redutases/genética , Neoplasias Cutâneas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Glutamina/metabolismo , Humanos , Masculino , Melanoma/patologia , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Neoplasias Cutâneas/patologia
4.
Medicine (Baltimore) ; 98(28): e16384, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31305441

RESUMO

Pyrroline-5-carboxylate reductase 1 (PYCR1) is an enzyme involved in cell metabolism and is upregulated in cancer. However, the correlations of PYCR1 expression with the clinicopathological features and prognosis of renal cell carcinoma (RCC) remain unclear. The purpose of this study was to identify the expression of PYCR1 and its clinical relevance in RCC patients.PYCR1 mRNA expression differences between RCC and the adjacent normal renal tissues were assessed using the Cancer Genome Atlas database (TCGA). Subsequently, the expression of PYCR1 mRNA and protein were evaluated by quantitative real-time polymerase chain reaction, Western blot, and immunochemistry using 30 paired frozen samples of RCC and the adjacent normal renal tissues. The protein expression of PYCR1 was evaluated by immunostaining formalin-fixed, paraffin-embedded sections of RCC samples from 96 patients who underwent radical nephrectomy, and its relationship with clinical features were analyzed. Nonpaired t tests were used to statistically analyze the differences between the 2 groups. Cox univariable and multivariable analyses of overall survival (OS) among RCC patients were performed.The expression of PYCR1 mRNA was significantly upregulated in RCC tissues compared to adjacent normal renal tissues in the TCGA database (P < .01). The area under the receiver operating characteristic curve value was 0.748. The expression of PYCR1 mRNA and protein was significantly upregulated in RCC compared with that in paired normal renal tissues (P < .01). Higher PYCR1 levels were associated with metastasis (P < .01). Kaplan-Meier survival curves indicated that higher PYCR1 expression was correlated with poorer OS. Therefore, PYCR1 may act as a novel prognostic marker and therapeutic target in the diagnosis and treatment of RCC.


Assuntos
Carcinoma de Células Renais/enzimologia , Neoplasias Renais/enzimologia , Pirrolina Carboxilato Redutases/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/enzimologia , Rim/patologia , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/metabolismo , Análise de Sobrevida
5.
Neoplasia ; 21(7): 665-675, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31108370

RESUMO

SIRT3 is a major mitochondrial deacetylase, which regulates various metabolic pathways by deacetylation; however, the effect of SIRT3 on proline metabolism is not reported. Pyrroline-5-carboxylate reductase 1 (PYCR1) participates in proline synthesis process by catalyzing the reduction of P5C to proline with concomitant generation of NAD+ and NADP+. PYCR1 is highly expressed in various cancers, and it can promote the growth of tumor cells. Here, through immunoprecipitation and mass spectrometry, we found that PYCR1 is in SIRT3's interacting network. PYCR1 directly binds to SIRT3 both in vivo and in vitro. CBP is the acetyltransferase for PYCR1, whereas SIRT3 deacetylates PYCR1. We further identified that K228 is the major acetylation site for PYCR1. Acetylation of PYCR1 at K228 reduced its enzymatic activity by impairing the formation of the decamer of PYCR1. As a result, acetylation of PYCR1 at K228 inhibits cell proliferation, while deacetylation of PYCR1 mediated by SIRT3 increases PYCR1's activity. Our findings on the regulation of PYCR1 linked proline metabolism with SIRT3, CBP and cell growth, thus providing a potential approach for cancer therapy.


Assuntos
Neoplasias/genética , Fragmentos de Peptídeos/genética , Pirrolina Carboxilato Redutases/genética , Sialoglicoproteínas/genética , Sirtuína 3/genética , Acetilação , Proliferação de Células/genética , Humanos , Células MCF-7 , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias/patologia , Prolina/biossíntese , Prolina/metabolismo
6.
Cells ; 8(5)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091804

RESUMO

Aging is a natural process that internal gene control and external stimuli mediate. Clinical data pointed out that homozygotic or heterozygotic mutation in the pyrroline-5-carboxylate reductase 1 (PYCR1) gene in humans caused cutis laxa (ARCL) disease, with progeroid appearance, lax and wrinkled skin, joint laxity, osteopenia, and mental retardation phenotypes. In this study, we aimed to generate pycr1 knockout (KO) zebrafish and carried out biochemical characterizations and behavior analyses. Marked apoptosis and senescence were detected in pycr1 KO zebrafish, which started from embryos/larvae stage. Biochemical assays showed that adult pycr1 KO fish have significantly reduced proline and extracellular matrix contents, lowered energy, and diminished superoxide dismutase (SOD) and telomerase activity when compared to the wild type fish, which suggested the pycr1 KO fish may have dysfunction in mitochondria. The pycr1 KO fish were viable; however, displayed progeria-like phenotype from the 4 months old and reach 50% mortality around six months old. In adult stage, we found that pycr1 KO fish showed reduced locomotion activity, aggression, predator avoidance, social interaction interest, as well as dysregulated color preference and circadian rhythm. In summary, we have identified multiple behavioral alterations in a novel fish model for aging with pycr1 gene loss-of-function by behavioral tests. This animal model may not only provide a unique vertebrate model to screen potential anti-aging drugs in the future, but also be an excellent in vivo model towards a better understanding of the corresponding behavioral alterations that accompany aging.


Assuntos
Envelhecimento/genética , Sintomas Comportamentais/genética , Modelos Animais de Doenças , Modelos Animais , Progéria/genética , Pirrolina Carboxilato Redutases/genética , Peixe-Zebra/genética , Envelhecimento/metabolismo , Animais , Metabolismo Energético , Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Locomoção , Mutação com Perda de Função , Mitocôndrias/metabolismo , Prolina/metabolismo , Pirrolina Carboxilato Redutases/fisiologia , Superóxido Dismutase/metabolismo
7.
Cancer Biother Radiopharm ; 34(6): 380-387, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30916574

RESUMO

Background: Pyrroline-5-carboxylate reductase 1 (PYCR1) is involved in tumor progression, for instance, breast cancer and prostate cancer. However, its role in tumor metastasis, especially in nonsmall cell lung cancer (NSCLC), is still elusive. Materials and Methods: The messenger RNA (mRNA) expression of PYCR1 between NSCLC and normal lung specimens was compared using Oncomine database. The endogenous PYCR1 expressions in NSCLC cell lines 95C and H1299 were knocked down by lentiviral-mediated delivery of short hairpin RNA (shRNA). Then the effects of PYCR1 on the migration and invasion of NSCLC cells were studied by wound healing assay and transwell assay. Results: PYCR1 mRNA expression was significantly higher in NSCLC specimens than that in normal lung tissues. Depletion of PYCR1 in NSCLC cell significantly repressed the cell migration and invasion. Moreover, depletion of PYCR1 influenced the expression of epithelial-mesenchymal transition molecules E-cadherin, Vimentin, N-cadherin, and Snail1. Conclusions: Our data suggested that PYCR1 plays a positive role in NSCLC metastasis in vitro and might be a promising target for treating NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/patologia , Pirrolina Carboxilato Redutases/metabolismo , Apoptose , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Invasividade Neoplásica , Pirrolina Carboxilato Redutases/genética , Células Tumorais Cultivadas
8.
Nat Commun ; 10(1): 845, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783087

RESUMO

Cell metabolism is strongly influenced by mechano-environment. We show here that a fraction of kindlin-2 localizes to mitochondria and interacts with pyrroline-5-carboxylate reductase 1 (PYCR1), a key enzyme for proline synthesis. Extracellular matrix (ECM) stiffening promotes kindlin-2 translocation into mitochondria and its interaction with PYCR1, resulting in elevation of PYCR1 level and consequent increase of proline synthesis and cell proliferation. Depletion of kindlin-2 reduces PYCR1 level, increases reactive oxygen species (ROS) production and apoptosis, and abolishes ECM stiffening-induced increase of proline synthesis and cell proliferation. In vivo, both kindlin-2 and PYCR1 levels are markedly increased in lung adenocarcinoma. Ablation of kindlin-2 in lung adenocarcinoma substantially reduces PYCR1 and proline levels, and diminishes fibrosis in vivo, resulting in marked inhibition of tumor growth and reduction of mortality rate. Our findings reveal a mechanoresponsive kindlin-2-PYCR1 complex that links mechano-environment to proline metabolism and signaling, and suggest a strategy to inhibit tumor growth.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Proteínas do Citoesqueleto/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Prolina/biossíntese , Células A549 , Adenocarcinoma de Pulmão/patologia , Animais , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Proteínas do Citoesqueleto/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/genética , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Proteínas de Neoplasias/genética , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismo
9.
Biomed Pharmacother ; 111: 588-595, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30605882

RESUMO

PYCR1 is over-expressed in non-small-cell lung cancer (NSCLC) and its high expression accelerates the progression of NSCLC. However, the underlying mechanisms of PYCR1 in NSCLC progression remain poorly understood. Our study determined the mechanisms of PYCR1 in promotion of the occurrence and development of NSCLC in vitro and in vivo. Firstly, the expression patterns of PYCR1 in NSCLC tissues and cells were determined by RT-PCR, western blot and immunohistochemistry. Then, the effects of PYCR1 on cell proliferation and apoptosis were evaluated by CCK-8 and flow cytomery assays. Finally, we explored the up-regulatory microRNAs (miRs) of PYCR1 and determined if MAPK pathway involved in this process. PYCR1 expression was elevated in NSCLC tissue samples and cells, and the high expression of PYCR1 closely associated with patients' advanced clinical process and poor outcome. Up-regulation of PYCR1 significantly increased the expression of p38 and promoted its nuclear accumulation. Besides, PYCR1 expression was negatively regulated by miR-488, and up-regulation of miR-488 significantly inhibited cell proliferation and tumorigenesis and increased cell apoptosis, and decreased p38 expression and its nuclear accumulation, whereas up-regulation of PYCR1 rescued these results induced by miR-488 over-expression. Collectively, these data suggest the mechanism of PYCR1 in promotion of NSCLC progression. PYCR1 is negatively regulated by miR-488 and then promotes the occurrence and development of NSCLC and activates p38 MAPK pathway.


Assuntos
Carcinogênese/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Progressão da Doença , Neoplasias Pulmonares/metabolismo , MicroRNAs/biossíntese , Pirrolina Carboxilato Redutases/biossíntese , Células A549 , Idoso , Animais , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Pirrolina Carboxilato Redutases/genética , Distribuição Aleatória , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Proc Natl Acad Sci U S A ; 116(1): 52-57, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30559182

RESUMO

Characterization of tumor metabolism with spatial information contributes to our understanding of complex cancer metabolic reprogramming, facilitating the discovery of potential metabolic vulnerabilities that might be targeted for tumor therapy. However, given the metabolic variability and flexibility of tumors, it is still challenging to characterize global metabolic alterations in heterogeneous cancer. Here, we propose a spatially resolved metabolomics approach to discover tumor-associated metabolites and metabolic enzymes directly in their native state. A variety of metabolites localized in different metabolic pathways were mapped by airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) in tissues from 256 esophageal cancer patients. In combination with in situ metabolomics analysis, this method provided clues into tumor-associated metabolic pathways, including proline biosynthesis, glutamine metabolism, uridine metabolism, histidine metabolism, fatty acid biosynthesis, and polyamine biosynthesis. Six abnormally expressed metabolic enzymes that are closely associated with the altered metabolic pathways were further discovered in esophageal squamous cell carcinoma (ESCC). Notably, pyrroline-5-carboxylate reductase 2 (PYCR2) and uridine phosphorylase 1 (UPase1) were found to be altered in ESCC. The spatially resolved metabolomics reveal what occurs in cancer at the molecular level, from metabolites to enzymes, and thus provide insights into the understanding of cancer metabolic reprogramming.


Assuntos
Metabolômica/métodos , Neoplasias/metabolismo , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Espectrometria de Massas , Neoplasias/enzimologia , Neoplasias/patologia , Pirrolina Carboxilato Redutases/metabolismo , Uridina Fosforilase/metabolismo
12.
Hum Genet ; 137(11-12): 921-939, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30450527

RESUMO

Juvenile segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ starting in childhood. Hutchinson-Gilford progeria syndrome (HGPS), caused by a recurrent de novo synonymous LMNA mutation resulting in aberrant splicing and generation of a mutant product called progerin, is a prototypical example of such disorders. Here, we performed a joint collaborative study using massively parallel sequencing and targeted Sanger sequencing, aimed at delineating the underlying genetic cause of 14 previously undiagnosed, clinically heterogeneous, non-LMNA-associated juvenile progeroid patients. The molecular diagnosis was achieved in 11 of 14 cases (~ 79%). Furthermore, we firmly establish biallelic mutations in POLR3A as the genetic cause of a recognizable, neonatal, Wiedemann-Rautenstrauch-like progeroid syndrome. Thus, we suggest that POLR3A mutations are causal for a portion of under-diagnosed early-onset segmental progeroid syndromes. We additionally expand the clinical spectrum associated with PYCR1 mutations by showing that they can somewhat resemble HGPS in the first year of life. Moreover, our results lead to clinical reclassification in one single case. Our data emphasize the complex genetic and clinical heterogeneity underlying progeroid disorders.


Assuntos
Retardo do Crescimento Fetal/genética , Progéria/genética , Pirrolina Carboxilato Redutases/genética , RNA Polimerase III/genética , Adolescente , Processamento Alternativo/genética , Criança , Feminino , Retardo do Crescimento Fetal/diagnóstico , Retardo do Crescimento Fetal/patologia , Predisposição Genética para Doença , Humanos , Lactente , Lamina Tipo A/genética , Masculino , Mutação , Fenótipo , Progéria/diagnóstico , Progéria/patologia , Progéria/fisiopatologia
13.
Biochemistry ; 57(25): 3433-3444, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29648801

RESUMO

Interest in how proline contributes to cancer biology is expanding because of the emerging role of a novel proline metabolic cycle in cancer cell survival, proliferation, and metastasis. Proline biosynthesis and degradation involve the shared intermediate Δ1-pyrroline-5-carboxylate (P5C), which forms l-glutamate-γ-semialdehyde (GSAL) in a reversible non-enzymatic reaction. Proline is synthesized from glutamate or ornithine through GSAL/P5C, which is reduced to proline by P5C reductase (PYCR) in a NAD(P)H-dependent reaction. The degradation of proline occurs in the mitochondrion and involves two oxidative steps catalyzed by proline dehydrogenase (PRODH) and GSAL dehydrogenase (GSALDH). PRODH is a flavin-dependent enzyme that couples proline oxidation with reduction of membrane-bound quinone, while GSALDH catalyzes the NAD+-dependent oxidation of GSAL to glutamate. PRODH and PYCR form a metabolic relationship known as the proline-P5C cycle, a novel pathway that impacts cellular growth and death pathways. The proline-P5C cycle has been implicated in supporting ATP production, protein and nucleotide synthesis, anaplerosis, and redox homeostasis in cancer cells. This Perspective details the structures and reaction mechanisms of PRODH and PYCR and the role of the proline-P5C cycle in cancer metabolism. A major challenge in the field is to discover inhibitors that specifically target PRODH and PYCR isoforms for use as tools for studying proline metabolism and the functions of the proline-P5C cycle in cancer. These molecular probes could also serve as lead compounds in cancer drug discovery targeting the proline-P5C cycle.


Assuntos
Neoplasias/metabolismo , Prolina/metabolismo , Animais , Vias Biossintéticas , Proliferação de Células , Humanos , Simulação de Acoplamento Molecular , Oxirredução , Prolina Oxidase/metabolismo , Pirrolina Carboxilato Redutases/metabolismo
14.
Cell Rep ; 22(12): 3107-3114, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29562167

RESUMO

Since the discovery of mutations in isocitrate dehydrogenase 1 (IDH1) in gliomas and other tumors, significant efforts have been made to gain a deeper understanding of the consequences of this oncogenic mutation. One aspect of the neomorphic function of the IDH1 R132H enzyme that has received less attention is the perturbation of cellular redox homeostasis. Here, we describe a biosynthetic pathway exhibited by cells expressing mutant IDH1. By virtue of a change in cellular redox homeostasis, IDH1-mutated cells synthesize excess glutamine-derived proline through enhanced activity of pyrroline 5-carboxylate reductase 1 (PYCR1), coupled to NADH oxidation. Enhanced proline biosynthesis partially uncouples the electron transport chain from tricarboxylic acid (TCA) cycle activity through the maintenance of a lower NADH/NAD+ ratio and subsequent reduction in oxygen consumption. Thus, we have uncovered a mechanism by which tumor cell survival may be promoted in conditions associated with perturbed redox homeostasis, as occurs in IDH1-mutated glioma.


Assuntos
Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Mutação , Prolina/biossíntese , Pirrolina Carboxilato Redutases/metabolismo , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Técnicas de Silenciamento de Genes , Glutamina/metabolismo , Homeostase , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Oligodendroglioma , Oxirredução , Pirrolina Carboxilato Redutases/genética
15.
Br J Cancer ; 118(2): 258-265, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29169183

RESUMO

BACKGROUND: Altered cellular metabolism is a hallmark of cancer and some are reliant on glutamine for sustained proliferation and survival. We hypothesise that the glutamine-proline regulatory axis has a key role in breast cancer (BC) in the highly proliferative classes. METHODS: Glutaminase (GLS), pyrroline-5-carboxylate synthetase (ALDH18A1), and pyrroline-5-carboxylate reductase 1 (PYCR1) were assessed at DNA/mRNA/protein levels in large, well-characterised cohorts. RESULTS: Gain of PYCR1 copy number and high PYCR1 mRNA was associated with Luminal B tumours. High ALDH18A1 and high GLS protein expression was observed in the oestrogen receptor (ER)+/human epidermal growth factor receptor (HER2)- high proliferation class (Luminal B) compared with ER+/HER2- low proliferation class (Luminal A) (P=0.030 and P=0.022 respectively), however this was not observed with mRNA. Cluster analysis of the glutamine-proline regulatory axis genes revealed significant associations with molecular subtypes of BC and patient outcome independent of standard clinicopathological parameters (P=0.012). High protein expression of the glutamine-proline enzymes were all associated with high MYC protein in Luminal B tumours only (P<0.001). CONCLUSIONS: We provide comprehensive clinical data indicating that the glutamine-proline regulatory axis plays an important role in the aggressive subclass of luminal BC and is therefore a potential therapeutic target.


Assuntos
Neoplasias da Mama/genética , Glutaminase/genética , Prolina/genética , Proteínas Proto-Oncogênicas c-myc/genética , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Neoplasias da Mama/metabolismo , Feminino , Dosagem de Genes , Redes Reguladoras de Genes , Genes myc , Glutaminase/metabolismo , Glutamina/genética , Glutamina/metabolismo , Humanos , Prolina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Environ Microbiol ; 19(9): 3700-3720, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28752945

RESUMO

The ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ-ProA-ProH), osmostress-adaptive synthesis of the compatible solute proline. The proA-encoded gamma-glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB-ProA-ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress-responsive proline production routes. A key player for enhanced osmostress-adaptive proline production is the osmotically regulated proHJ promoter. We used site-directed mutagenesis to study the salient features of this stress-responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA-type promoters serve to keep transcription low under non-inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA-type promoters of either 16-bp (the natural proHJ promoter), or 18-bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress-inducibility of transcription.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Prolina/biossíntese , Pirrolina Carboxilato Redutases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Glutamato-5-Semialdeído Desidrogenase/genética , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Mutação Puntual/genética , Regiões Promotoras Genéticas/genética
17.
Plant Sci ; 262: 39-51, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28716419

RESUMO

ζ-Carotene desaturase (ZDS) is one of the key enzymes in carotenoid biosynthesis pathway. However, the ZDS gene has not been applied to carotenoid improvement of plants. Its roles in tolerance to abiotic stresses have not been reported. In this study, the IbZDS gene was isolated from storage roots of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Nongdafu 14. Its overexpression significantly increased ß-carotene and lutein contents and enhanced salt tolerance in transgenic sweetpotato (cv. Kokei No. 14) plants. Significant up-regulation of lycopene ß-cyclase (ß-LCY) and ß-carotene hydroxylase (ß-CHY) genes and significant down-regulation of lycopene ε-cyclase (ε-LCY) and ε-carotene hydroxylase (ε-CHY) genes were found in the transgenic plants. Abscisic acid (ABA) and proline contents and superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants under salt stress. The salt stress-responsive genes encoding pyrroline-5-carboxylate reductase (P5CR), SOD, CAT, ascorbate peroxidase (APX) and POD were found to be significantly up-regulated in the transgenic plants under salt stress. This study indicates that the IbZDS gene has the potential to be applied for improving ß-carotene and lutein contents and salt tolerance in sweetpotato and other plants.


Assuntos
Ipomoea batatas/enzimologia , Ipomoea batatas/metabolismo , Luteína/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/metabolismo , beta Caroteno/metabolismo , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Catalase/genética , Catalase/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Ipomoea batatas/efeitos dos fármacos , Peroxidase/genética , Peroxidase/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismo , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
18.
Nat Commun ; 8: 15267, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28492237

RESUMO

Metastases are the leading cause of mortality in patients with cancer. Metastasis formation requires cancer cells to adapt their cellular phenotype. However, how metabolism supports this adaptation of cancer cells is poorly defined. We use 2D versus 3D cultivation to induce a shift in the cellular phenotype of breast cancer cells. We discover that proline catabolism via proline dehydrogenase (Prodh) supports growth of breast cancer cells in 3D culture. Subsequently, we link proline catabolism to in vivo metastasis formation. In particular, we find that PRODH expression and proline catabolism is increased in metastases compared to primary breast cancers of patients and mice. Moreover, inhibiting Prodh is sufficient to impair formation of lung metastases in the orthotopic 4T1 and EMT6.5 mouse models, without adverse effects on healthy tissue and organ function. In conclusion, we discover that Prodh is a potential drug target for inhibiting metastasis formation.


Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Prolina/metabolismo , Trifosfato de Adenosina , Aldeído Desidrogenase/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos C57BL , Prolina Oxidase/metabolismo , Pirrolina Carboxilato Redutases , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
20.
Carcinogenesis ; 38(5): 519-531, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28379297

RESUMO

Human mitochondrial pyrroline-5-carboxylate reductase (PYCR) is a house-keeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate to proline. This enzymatic cycle plays pivotal roles in amino acid metabolism, intracellular redox potential and mitochondrial integrity. Here, we hypothesize that PYCR1 might be a novel prognostic biomarker and therapeutic target for breast cancer. In this study, breast cancer tissue samples were obtained from Zhejiang University (ZJU set). Immunohistochemistry analysis was performed to detect the protein level of PYCR1, and Kaplan-Meier and Cox proportional analyses were employed in this outcome study. The prognostic significance and performance of PYCR1 mRNA were validated on 13 worldwide independent microarray data sets, composed of 2500 assessable breast cancer cases. Our findings revealed that both PYCR1 mRNA and protein expression were significantly associated with tumor size, grade and invasive molecular subtypes of breast cancers. Independent and pooled analyses verified that higher PYCR1 mRNA levels were significantly associated with poor survival of breast cancer patients, regardless of estrogen receptor (ER) status. For in vitro studies, inhibition of PYCR1 by small-hairpin RNA significantly reduced the growth and invasion capabilities of the cells, while enhancing the cytotoxicity of doxorubicin in breast cancer cell lines MCF-7 (ER positive) and MDA-MB-231 (ER negative). Further population study also validated that chemotherapy significantly improved survival in early-stage breast cancer patients with low PYCR1 expression levels. Therefore, PYCR1 might serve as a prognostic biomaker for either ER-positive or ER-negative breast cancer subtypes and can also be a potential target for breast cancer therapy.


Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Mitocôndrias/genética , Pirrolina Carboxilato Redutases/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Análise por Conglomerados , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Mitocôndrias/metabolismo , Gradação de Tumores , Invasividade Neoplásica , Fenótipo , Prognóstico , Pirrolina Carboxilato Redutases/metabolismo , Receptores Estrogênicos/metabolismo
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