RESUMO
BACKGROUND AND AIMS: Methionine adenosyltransferase alpha1 (MATα1) is responsible for the biosynthesis of S-adenosylmethionine in normal liver. Alcohol consumption enhances MATα1 interaction with peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), which blocks MATα1 mitochondrial targeting, resulting in lower mitochondrial MATα1 content and mitochondrial dysfunction in alcohol-associated liver disease (ALD) in part through upregulation of cytochrome P450 2E1. Conversely, alcohol intake enhances SUMOylation, which enhances cytochrome P450 2E1 expression. MATα1 has potential SUMOylation sites, but whether MATα1 is regulated by SUMOylation in ALD is unknown. Here, we investigated if MATα1 is regulated by SUMOylation and, if so, how it impacts mitochondrial function in ALD. APPROACH AND RESULTS: Proteomics profiling revealed hyper-SUMOylation of MATα1, and prediction software identified lysine 48 (K48) as the potential SUMOylation site in mice (K47 in humans). Experiments with primary hepatocytes, mouse, and human livers revealed that SUMOylation of MAT1α by SUMO2 depleted mitochondrial MATα1. Furthermore, mutation of MATα1 K48 prevented ethanol-induced mitochondrial membrane depolarization, MATα1 depletion, and triglyceride accumulation. Additionally, CRISPR/CRISPR associated protein 9 gene editing of MATα1 at K48 hindered ethanol-induced MATα1-PIN1 interaction, degradation, and phosphorylation of MATα1 in vitro. In vivo, CRISPR/CRISPR associated protein 9 MATα1 K48 gene-edited mice were protected from ethanol-induced fat accumulation, liver injury, MATα1-PIN1 interaction, mitochondrial MATα1 depletion, mitochondrial dysfunction, and low S-adenosylmethionine levels. CONCLUSIONS: Taken together, our findings demonstrate an essential role for SUMOylation of MATα1 K48 for interaction with PIN1 in ALD. Preventing MATα1 K48 SUMOylation may represent a potential treatment strategy for ALD.
Assuntos
Hepatopatias Alcoólicas , Metionina Adenosiltransferase , Sumoilação , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/genética , Animais , Camundongos , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/genética , Humanos , Mitocôndrias Hepáticas/metabolismo , Masculino , Hepatócitos/metabolismo , Fígado/metabolismoRESUMO
BACKGROUND & AIMS: The liver is a common site of cancer metastasis, most commonly from colorectal cancer, and primary liver cancers that have metastasized are associated with poor outcomes. The underlying mechanisms by which the liver defends against these processes are largely unknown. Prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) are highly expressed in the liver. They positively regulate each other and their deletion results in primary liver cancer. Here we investigated their roles in primary and secondary liver cancer metastasis. METHODS: We identified common target genes of PHB1 and MAT1A using a metastasis array, and measured promoter activity and transcription factor binding using luciferase reporter assays and chromatin immunoprecipitation, respectively. We examined how PHB1 or MAT1A loss promotes liver cancer metastasis and whether their loss sensitizes to colorectal liver metastasis (CRLM). RESULTS: Matrix metalloproteinase-7 (MMP-7) is a common target of MAT1A and PHB1 and its induction is responsible for increased migration and invasion when MAT1A or PHB1 is silenced. Mechanistically, PHB1 and MAT1A negatively regulate MMP7 promoter activity via an AP-1 site by repressing the MAFG-FOSB complex. Loss of MAT1A or PHB1 also increased MMP-7 in extracellular vesicles, which were internalized by colon and pancreatic cancer cells to enhance their oncogenicity. Low hepatic MAT1A or PHB1 expression sensitized to CRLM, but not if endogenous hepatic MMP-7 was knocked down first, which lowered CD4+ T cells while increasing CD8+ T cells in the tumor microenvironment. Hepatocytes co-cultured with colorectal cancer cells express less MAT1A/PHB1 but more MMP-7. Consistently, CRLM raised distant hepatocytes' MMP-7 expression in mice and humans. CONCLUSION: We have identified a PHB1/MAT1A-MAFG/FOSB-MMP-7 axis that controls primary liver cancer metastasis and sensitization to CRLM. IMPACT AND IMPLICATIONS: Primary and secondary liver cancer metastasis is associated with poor outcomes but whether the liver has underlying defense mechanism(s) against metastasis is unknown. Here we examined the hypothesis that hepatic prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) cooperate to defend the liver against metastasis. Our studies found PHB1 and MAT1A form a complex that suppresses matrix metalloproteinase-7 (MMP-7) at the transcriptional level and loss of either PHB1 or MAT1A sensitizes the liver to metastasis via MMP-7 induction. Strategies that target the PHB1/MAT1A-MMP-7 axis may be a promising approach for the treatment of primary and secondary liver cancer metastasis.
Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/metabolismo , Neoplasias Colorretais/genética , Neoplasias Hepáticas/patologia , Metaloproteinase 7 da Matriz/genética , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Proibitinas , Microambiente TumoralRESUMO
BACKGROUND AND AIMS: The sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC. APPROACH AND RESULTS: Tissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations ( E pCAM + CD63 + , C D147 + CD63 + , and G PC3 + CD63 + HCC EVs), was established for detecting early-stage HCC. A phase 2 biomarker study was conducted to evaluate the performance of ECG score in a training cohort ( n = 106) and an independent validation cohort ( n = 72).Overall, 99.7% of tissue microarray stained positive for at least one of the four HCC-associated protein markers (EpCAM, CD147, GPC3, and ASGPR1) that were subsequently validated in HCC EVs. In the training cohort, HCC EV ECG score demonstrated an area under the receiver operating curve (AUROC) of 0.95 (95% confidence interval [CI], 0.90-0.99) for distinguishing early-stage HCC from cirrhosis with a sensitivity of 91% and a specificity of 90%. The AUROCs of the HCC EV ECG score remained excellent in the validation cohort (0.93; 95% CI, 0.87-0.99) and in the subgroups by etiology (viral: 0.95; 95% CI, 0.90-1.00; nonviral: 0.94; 95% CI, 0.88-0.99). CONCLUSION: HCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.
Assuntos
Carcinoma Hepatocelular , Vesículas Extracelulares , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Biomarcadores Tumorais/análise , Vesículas Extracelulares/química , Proteínas de Membrana , Eletrocardiografia , GlipicanasRESUMO
Methionine adenosyltransferase α1 (MATα1, encoded by MAT1A) is responsible for hepatic biosynthesis of S-adenosyl methionine, the principal methyl donor. MATα1 also act as a transcriptional cofactor by interacting and influencing the activity of several transcription factors. Mat1a knockout (KO) mice have increased levels of cytochrome P450 2E1 (CYP2E1), but the underlying mechanisms are unknown. The aims of the current study were to identify binding partners of MATα1 and elucidate how MATα1 regulates CYP2E1 expression. We identified binding partners of MATα1 by coimmunoprecipitation (co-IP) and mass spectrometry. Interacting proteins were confirmed using co-IP using recombinant proteins, liver lysates, and mitochondria. Alcoholic liver disease (ALD) samples were used to confirm relevance of our findings. We found that MATα1 negatively regulates CYP2E1 at mRNA and protein levels, with the latter being the dominant mechanism. MATα1 interacts with many proteins but with a predominance of mitochondrial proteins including CYP2E1. We found that MATα1 is present in the mitochondrial matrix of hepatocytes using immunogold electron microscopy. Mat1a KO hepatocytes had reduced mitochondrial membrane potential and higher mitochondrial reactive oxygen species, both of which were normalized when MAT1A was overexpressed. In addition, KO hepatocytes were sensitized to ethanol and tumor necrosis factor α-induced mitochondrial dysfunction. Interaction of MATα1 with CYP2E1 was direct, and this facilitated CYP2E1 methylation at R379, leading to its degradation through the proteasomal pathway. Mat1a KO livers have a reduced methylated/total CYP2E1 ratio. MATα1's influence on mitochondrial function is largely mediated by its effect on CYP2E1 expression. Patients with ALD have reduced MATα1 levels and a decrease in methylated/total CYP2E1 ratio. Conclusion: Our findings highlight a critical role of MATα1 in regulating mitochondrial function by suppressing CYP2E1 expression at multiple levels.
Assuntos
Citocromo P-450 CYP2E1/genética , Metionina Adenosiltransferase/fisiologia , Mitocôndrias Hepáticas/fisiologia , Animais , Feminino , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Hepatopatias Alcoólicas/metabolismo , Masculino , Potencial da Membrana Mitocondrial , Metilação , Camundongos , Proteínas Mitocondriais/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Alcoholic liver injury is associated with hepatic stellate cell (HSC) activation. A-kinase anchoring protein 12 (AKAP12) scaffolds protein kinase C and cyclin-D1, which is regulated by its phosphorylation, and spatiotemporally controls cell proliferation, invasiveness, and chemotaxis. HSC activation induces AKAP12 expression, but the role of AKAP12's scaffolding activity in liver function is unknown. Because AKAP12 phosphorylation is enhanced in ethanol-treated HSCs, we examined AKAP12's scaffolding functions in alcohol-mediated HSC activation and liver injury. AKAP12 expression, interaction, and phosphorylation were assayed in in vitro and in vivo ethanol models and human subjects by real-time PCR, coimmunoprecipitation, immunoblotting, and phosphorylated proteomics/Phos-tag. Ethanol induced AKAP12 phosphorylation in the liver and in primary HSCs, but not in hepatocytes. AKAP12's scaffolding activity for protein kinase C/cyclin-D1 decreased in ethanol-treated HSCs but not hepatocytes. AKAP12 negatively regulated HSC activation, which was reversed by ethanol-mediated AKAP12 phosphorylation. AKAP12 interacted with heat shock protein 47 (HSP47), which chaperones collagen and induces its secretion. Ethanol inhibited AKAP12-HSP47 and induced HSP47-collagen interaction. Ethanol-induced phosphorylated AKAP12 was unable to bind to HSP47 compared with its unphosphorylated counterpart, thereby proving that ethanol-mediated phosphorylation of AKAP12 inhibited the HSP47-AKAP12 scaffold. Silencing AKAP12 facilitated the chaperoning of collagen by HSP47. Hence, AKAP12 scaffolds HSP47 and regulates collagen-HSP47 interaction. Ethanol quenches AKAP12's scaffolding activity through phosphorylation and facilitates HSC activation.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Estreladas do Fígado/metabolismo , Hepatopatias Alcoólicas/metabolismo , Fígado/metabolismo , Animais , Colágeno/metabolismo , Ciclina D1/metabolismo , Etanol/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Hepatopatias Alcoólicas/patologia , Camundongos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismoRESUMO
Alcohol acts through numerous pathways leading to alcoholic liver disease (ALD). Cytochrome P450 (CYP2E1), an ethanol-inducible enzyme, metabolizes ethanol-producing toxic reactive oxygen species (ROS) and is regulated at the posttranslational level. Small ubiquitin-like modifier (SUMO)ylation is a posttranslational modification that involves the addition of SUMOs, which modulate protein stability, activity, and localization. We demonstrated that ubiquitin-conjugation enzyme 9, the SUMO-conjugating enzyme, is induced in the livers of an intragastric ethanol mouse model. Our aim is to examine whether SUMOylation could regulate ethanol-induced CYP2E1 expression in ALD and to elucidate the molecular mechanism(s). CYP2E1 and UBC9 expression in vitro and in vivo was detected by real-time PCR and immunoblotting/immunostaining. SUMOylation was assayed by mass spectrometry and coimmunoprecipitation. Ubc9 expression was induced in ethanol-fed mouse livers, and silencing inhibited ethanol-mediated CYP2E1 microsomal retention and enzymatic activity. CYP2E1 SUMOylation was found to be induced by ethanol in vitro and in vivo. Ubc9 silencing prevents ethanol-induced lipid accumulation and ROS production. UBC9 was highly expressed in human ALD livers. Finally, we found that lysine 410 is a key SUMOylated residue contributing to CYP2E1 protein stability and activity preventing CYP2E1 SUMOylation. Ethanol-mediated up-regulation of CYP2E1 via SUMOylation enhancing its protein stability and activity and may have important implications in ALD.-Tomasi, M. L., Ramani, K., Ryoo, M., Cossu, C., Floris, A., Murray, B. J., Iglesias-Ara, A., Spissu, Y., Mavila, N. SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.
Assuntos
Citocromo P-450 CYP2E1/biossíntese , Etanol/efeitos adversos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatopatias Alcoólicas/enzimologia , Sumoilação/efeitos dos fármacos , Animais , Estabilidade Enzimática/efeitos dos fármacos , Etanol/farmacologia , Hepatopatias Alcoólicas/patologia , Camundongos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/patologia , Espécies Reativas de Oxigênio/metabolismo , Enzimas de Conjugação de Ubiquitina/biossínteseRESUMO
Lipopolysaccharide (LPS), a bacterial endotoxin, induces inflammation in macrophages via activation of NF-κB signaling. Sumoylation is a post-translational modification mediated by the small ubiquitin-like modifier, SUMO. Ubiquitin-conjugating enzyme 9 (UBC9) is the only known SUMO conjugating enzyme. LPS treatment lowers SUMO-1 and UBC9 mRNA levels in primary astrocytes. UBC9 can degrade NF-κB inhibitor α (Ikbα) via a SUMO2/3-ubiquitin pathway. However, UBC9 may also promote Ikbα stability by SUMO-1 conjugation that further regulates NF-κB signaling. The role of UBC9 in liver inflammation is unknown. We reported that CDK1-mediated phosphorylation of UBC9 enhanced its stability. Herein, we describe an anti-inflammatory role of UBC9 that is lost when it is phosphorylated during inflammation. LPS exposure caused induction in UBC9 phosphorylation and CDK1 activation specifically in Kupffer cells in vivo and in RAW264.7 macrophages in vitro. Silencing or overexpression experiments in vitro and in vivo showed that UBC9 was required to blunt the proinflammatory response elicited by LPS. LPS stimulation raised the binding of phospho-UBC9 but not the unphosphorylated counterpart, to Ikbα in RAW264.7 macrophages. Hence, phospho-UBC9 may promote NF-κB signaling by regulating Ikbα and this may be a novel mechanism that deregulates liver inflammatory signaling.
Assuntos
Hepatite/metabolismo , Inflamação/metabolismo , Transdução de Sinais/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Western Blotting , Endotoxinas/toxicidade , Ensaio de Imunoadsorção Enzimática , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fosforilação , Reação em Cadeia da Polimerase , Células RAW 264.7 , TransfecçãoRESUMO
Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATß proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATß proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATß proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATß predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members (ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 [B-Raf], MEK). Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATß but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATß phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation.
Assuntos
Transdiferenciação Celular , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/enzimologia , Metionina Adenosiltransferase/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Estabilidade Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inativação Gênica , Humanos , Metionina Adenosiltransferase/química , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese/genética , Mutação/genética , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas B-raf/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND & AIMS: Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear. METHODS: By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients. RESULTS: Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not ß-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer. CONCLUSIONS: The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Carcinoma Hepatocelular/genética , Proteínas de Ligação a DNA/fisiologia , Hepatócitos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neoplasias Hepáticas/genética , Proteínas de Membrana/fisiologia , Proteínas Musculares/fisiologia , Fosfoproteínas/fisiologia , Receptores Notch/fisiologia , Fatores de Transcrição/fisiologia , Animais , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Jagged-1 , Neoplasias Hepáticas/metabolismo , Camundongos , Proteínas Serrate-Jagged , Fatores de Transcrição de Domínio TEA , Regulação para Cima , Proteínas de Sinalização YAPRESUMO
UNLABELLED: Methionine adenosyltransferase 2B (MAT2B) encodes for two variant proteins (V1 and V2) that promote cell growth. Using in-solution proteomics, GIT1 (G Protein Coupled Receptor Kinase Interacting ArfGAP 1) was identified as a potential interacting partner of MAT2B. Here, we examined the functional significance of this interplay. Coimmunoprecipitation experiments examined protein interactions. Tissue expression levels of proteins were examined using immunohistochemistry and western blotting. Expression levels of proteins were varied using transient knockdown or overexpression to observe the effect of alterations in each protein on the entire complex. Direct interaction among individual proteins was further verified using in vitro translated and recombinant proteins. We found both MAT2B variants interact with GIT1. Overexpression of V1, V2, or GIT1 activated mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-regulated kinase (ERK), raised cyclin D1 protein level, and increased growth, whereas the opposite occurred when V1, V2, or GIT1 was knocked down. MAT2B and GIT1 require each other to activate MEK1/ERK and increase growth. MAT2B directly interacts with MEK1, GIT1, and ERK2. Expression level of V1, V2, or GIT1 directly influenced recruitment of GIT1 or MAT2B and ERK2 to MEK1, respectively. In pull-down assays, MAT2B directly promoted binding of GIT1 and ERK2 to MEK1. MAT2B and GIT1 interact and are overexpressed in most human liver and colon cancer specimens. Increased expression of V1, V2, or GIT1 promoted growth in an orthotopic liver cancer model, whereas increased expression of either V1 or V2 with GIT1 further enhanced growth and lung metastasis. CONCLUSION: MAT2B and GIT1 form a scaffold, which recruits and activates MEK and ERK to promote growth and tumorigenesis. This novel MAT2B/GIT1 complex may provide a potential therapeutic gateway in human liver and colon cancer. (HEPATOLOGY 2012).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases , Metionina Adenosiltransferase/metabolismo , Processamento Alternativo , Transformação Celular Neoplásica , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Hep G2 , Humanos , Isoenzimas/metabolismo , MAP Quinase Quinase 1/metabolismo , Metástase NeoplásicaRESUMO
Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens, whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70-75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell.
Assuntos
Neoplasias do Colo/metabolismo , Neoplasias Hepáticas/metabolismo , Metionina Adenosiltransferase/metabolismo , Putrescina/metabolismo , Idoso , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/enzimologia , Feminino , Humanos , Neoplasias Hepáticas/enzimologia , Masculino , Metionina Adenosiltransferase/genética , Pessoa de Meia-Idade , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Interferente Pequeno , S-Adenosilmetionina/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição GênicaRESUMO
Endocrine resistance poses a significant clinical challenge for patients with hormone receptor-positive and human epithelial growth factor receptor 2-negative (HR + HER2-) breast cancer. Dysregulation of estrogen receptor (ER) and ERBB signaling pathways is implicated in resistance development; however, the integration of these pathways remains unclear. While SMAD4 is known to play diverse roles in tumorigenesis, its involvement in endocrine resistance is poorly understood. Here, we investigate the role of SMAD4 in acquired endocrine resistance in HR + HER2- breast cancer. Genome-wide CRISPR screening identifies SMAD4 as a regulator of 4-hydroxytamoxifen (OHT) sensitivity in T47D cells. Clinical data analysis reveals downregulated SMAD4 expression in breast cancer tissues, correlating with poor prognosis. Following endocrine therapy, SMAD4 expression is further suppressed. Functional studies demonstrate that SMAD4 depletion induces endocrine resistance in vitro and in vivo by enhancing ER and ERBB signaling. Concomitant inhibition of ER and ERBB signaling leads to aberrant autophagy activation. Simultaneous inhibition of ER, ERBB, and autophagy pathways synergistically impacts SMAD4-depleted cells. Our findings unveil a mechanism whereby endocrine therapy-induced SMAD4 downregulation drives acquired resistance by integrating ER and ERBB signaling and suggest a rational treatment strategy for endocrine-resistant HR + HER2- breast cancer patients.
Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Receptor ErbB-2 , Receptores de Estrogênio , Transdução de Sinais , Proteína Smad4 , Humanos , Proteína Smad4/metabolismo , Proteína Smad4/genética , Feminino , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Receptores de Estrogênio/metabolismo , Linhagem Celular Tumoral , Animais , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Tamoxifeno/análogos & derivados , Camundongos , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Camundongos Nus , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Receptores ErbB/metabolismo , Receptores ErbB/genéticaRESUMO
GSH is synthesized sequentially by glutamate-cysteine ligase (GCL) and GSH synthase and defends against oxidative stress, which promotes hepatic stellate cell (HSC) activation. Changes in GSH synthesis during HSC activation are poorly characterized. Here, we examined the expression of GSH synthetic enzymes in rat HSC activation and reversion to quiescence. Expression of the GCL catalytic subunit (GCLC) fell during HSC activation and increased when activated HSCs revert back to quiescence. Blocking the increase in GCLC expression kept HSCs in an activated state. Activated HSCs have higher nuclear levels and binding activity of MafG to the antioxidant response element (ARE) of GCLC but lower Nrf2/MafG heterodimer binding to the ARE. Quiescent HSCs have a lower nuclear MafG level but higher Nrf2/MafG heterodimer binding to ARE. This occurred because of enhanced sumoylation of Nrf2 and MafG by SUMO-1, which promoted Nrf2 binding to ARE and heterodimerization with MafG. In vivo, knockdown of GCLC exacerbated bile duct ligation-induced liver injury and fibrosis. Ursodeoxycholic acid and S-adenosylmethionine are anti-fibrotic in bile duct ligation, but this effect was nearly lost if GCLC induction was blocked. In conclusion, sumoylation of Nrf2 and MafG enhances heterodimerization and increases GCLC expression, which keeps HSCs in a quiescent state. Antifibrotic agents require activation of GCLC to fully exert their protective effect.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glutamato-Cisteína Ligase/biossíntese , Células Estreladas do Fígado/enzimologia , Cirrose Hepática/enzimologia , Animais , Sequência de Bases , Células Cultivadas , Ativação Enzimática , Técnicas de Silenciamento de Genes , Glutamato-Cisteína Ligase/genética , Células Estreladas do Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Fator de Transcrição MafG/genética , Fator de Transcrição MafG/metabolismo , Dados de Sequência Molecular , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta/genética , S-Adenosilmetionina/genética , S-Adenosilmetionina/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Sumoilação/genética , Ácido Ursodesoxicólico/genética , Ácido Ursodesoxicólico/metabolismoRESUMO
UNLABELLED: Methionine adenosyltransferases (MATs) are critical enzymes that catalyze the formation of the methyl donor S-adenosyl methionine (SAM). The MAT2A gene, which encodes the catalytic subunit α2, is induced in dedifferentiated liver. We previously demonstrated that MAT2A expression is enhanced in activated hepatic stellate cells (HSCs) and that silencing this gene reduces HSC activation. In this study, we examined the molecular mechanisms responsible for the transcriptional regulation of the MAT2A gene in HSCs. We identified peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) in the rat MAT2A promoter. The PPARγ agonist rosiglitazone (RSG) promoted quiescence in the activated rat HSC cell line (BSC) or culture-activated primary rat HSCs, decreased MAT2A expression and promoter activity, and enhanced PPARγ binding to MAT2A PPREs. In vivo HSC activation in bile duct-ligated rats lowered PPARγ interaction with MAT2A PPREs. Silencing PPARγ increased MAT2A transcription, whereas overexpressing it had the opposite effect, demonstrating that PPARγ negatively controls this gene. Site-directed mutagenesis of PPREs abolished PPARγ recruitment to the MAT2A promoter and its inhibitory effect on MAT2A transcription in quiescent HSCs. PPRE mutations decreased the basal promoter activity of MAT2A in activated HSCs independent of PPARγ, indicating that other factors might be involved in PPRE interaction. We identified PPARß binding to wild-type but not to mutated PPREs in activated cells. Furthermore, silencing PPARß inhibited MAT2A expression and promoter activity. Forced expression of MAT2A in RSG-treated HSCs lowered PPARγ and enhanced PPARß expression, thereby promoting an activated phenotype. CONCLUSION: We identified PPARγ as a negative regulator of MAT2A in quiescent HSCs. A switch from quiescence to activation abolishes this control and allows PPARß to up-regulate MAT2A transcription.
Assuntos
Células Estreladas do Fígado/metabolismo , Metionina Adenosiltransferase/genética , PPAR gama/fisiologia , PPAR beta/fisiologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Masculino , Metionina Adenosiltransferase/fisiologia , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Elementos de Resposta/fisiologia , Rosiglitazona , Tiazolidinedionas/farmacologia , Transcrição GênicaRESUMO
UNLABELLED: Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and is overexpressed in several malignancies, but its expression in hepatocellular carcinoma (HCC) is unknown. Hepatic S-adenosyl methionine (SAMe) levels decrease in methionine adenosyltransferase 1A (Mat1a) knockout (KO) mice, which develop HCC, and in ethanol-fed mice. We examined the regulation of Ubc9 by SAMe in murine liver and human HCC, breast, and colon carcinoma cell lines and specimens. Real-time polymerase chain reaction and western blotting measured gene and protein expression, respectively. Immunoprecipitation followed by western blotting examined protein-protein interactions. Ubc9 expression increased in HCC and when hepatic SAMe levels decreased. SAMe treatment in Mat1a KO mice reduced Ubc9 protein, but not messenger RNA (mRNA) levels, and lowered sumoylation. Similarly, treatment of liver cancer cell lines HepG2 and Huh7, colon cancer cell line RKO, and breast cancer cell line MCF-7 with SAMe or its metabolite 5'-methylthioadenosine (MTA) reduced only Ubc9 protein level. Ubc9 posttranslational regulation is unknown. Ubc9 sequence predicted a possible phosphorylation site by cell division cycle 2 (Cdc2), which directly phosphorylated recombinant Ubc9. Mat1a KO mice had higher phosphorylated (phospho)-Ubc9 levels, which normalized after SAMe treatment. SAMe and MTA treatment lowered Cdc2 mRNA and protein levels, as well as phospho-Ubc9 and protein sumoylation in liver, colon, and breast cancer cells. Serine 71 of Ubc9 was required for phosphorylation, interaction with Cdc2, and protein stability. Cdc2, Ubc9, and phospho-Ubc9 levels increased in human liver, breast, and colon cancers. CONCLUSION: Cdc2 expression is increased and Ubc9 is hyperphosphorylated in several cancers, and this represents a novel mechanism to maintain high Ubc9 protein expression that can be inhibited by SAMe and MTA.
Assuntos
Neoplasias Hepáticas/metabolismo , S-Adenosilmetionina/fisiologia , Sumoilação , Enzimas de Conjugação de Ubiquitina/biossíntese , Animais , Linhagem Celular Tumoral , Humanos , CamundongosRESUMO
Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT). MAT1A is silenced in hepatocellular carcinoma (HCC), and absence of MAT1A leads to spontaneous development of HCC in mice. Previous report correlated promoter methylation to silencing of MAT1A but definitive proof was lacking. Here we investigated the role of methylation in regulating MAT1A expression. There are three MspI/HpaII sites from -1,913 to +160 of the human MAT1A gene (numbered relative to the translational start site) at position -977, +10, and +88. Bisulfite treatment and DNA sequencing, and Southern blot analysis showed that methylation at +10 and +88, but not -977, correlated with lack of MAT1A expression. MAT1A promoter construct methylated at -977, +10 or +88 position has 0.7-fold, 3-fold, and 1.6-fold lower promoter activity, respectively. Methylation at -977 and +10 did not inhibit the promoter more than methylation at +10 alone; while methylation at +10 and +88 reduced promoter activity by 60%. Mutation of +10 and +88 sites also resulted in 40% reduction of promoter activity. Reactivation of MAT1A correlated with demethylation of +10 and +88. In vitro transcription assay showed that methylation or mutation of +10 and +88 sites reduced transcription. In conclusion, our data support the novel finding that methylation of the MAT1A coding region can inhibit gene transcription. This represents a key mechanism for decreased MAT1A expression in HCC and a target for therapy. To our knowledge, this is the first example of coding region methylation inhibiting transcription of a mammalian gene.
Assuntos
Metilação de DNA/genética , Metionina Adenosiltransferase/genética , Fases de Leitura Aberta , Sequência de Bases , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Células Cultivadas , DNA de Neoplasias/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas , Transcrição Gênica , TransfecçãoRESUMO
Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12's scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work, we demonstrate that AKAP12's scaffolding activity toward the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12's site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12's phospho-sites restores its scaffolding toward HSP47, inhibiting HSP47's collagen maturation functions, and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling, and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.
Assuntos
Proteínas de Ancoragem à Quinase A , Células Estreladas do Fígado , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Proteínas de Ciclo Celular , Colágeno/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclinas/metabolismo , Modelos Animais de Doenças , Fibrose , Proteínas de Choque Térmico HSP47/genética , Proteínas de Choque Térmico HSP47/metabolismo , Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Fosforilação , Proteína Quinase C/metabolismoRESUMO
UNLABELLED: Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis. CONCLUSION: Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes.
Assuntos
Carcinoma Hepatocelular/patologia , Cirrose Hepática/etiologia , Neoplasias Hepáticas/patologia , Proteínas Repressoras/fisiologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Knockout , Proibitinas , Proteínas Repressoras/deficiênciaRESUMO
UNLABELLED: Increased mitogen-activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2) and the dual-specificity MAPK phosphatase MKP-1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S-adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin-like and caspase-like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin-like and caspase-like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. CONCLUSION: DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration.
Assuntos
Fosfatase 1 de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hepatócitos/enzimologia , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/metabolismo , Animais , Humanos , Masculino , Metionina Adenosiltransferase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor downregulated in hepatocellular carcinoma and cholangiocarcinoma, two of the fastest rising cancers worldwide. We compared MATα1 (protein encoded by MAT1A) interactome in normal versus cancerous livers by mass spectrometry to reveal interactions with 14-3-3ζ. The MATα1/14-3-3ζ complex was critical for the expression of 14-3-3ζ. Similarly, the knockdown and small molecule inhibitor for 14-3-3ζ (BV02), and ChIP analysis demonstrated the role of 14-3-3ζ in suppressing MAT1A expression. Interaction between MATα1 and 14-3-3ζ occurs directly and is enhanced by AKT2 phosphorylation of MATα1. Blocking their interaction enabled nuclear MATα1 translocation and inhibited tumorigenesis. In contrast, overexpressing 14-3-3ζ lowered nuclear MATα1 levels and promoted tumor progression. However, tumor-promoting effects of 14-3-3ζ were eliminated when liver cancer cells expressed mutant MATα1 unable to interact with 14-3-3ζ. Taken together, the reciprocal negative regulation that MATα1 and 14-3-3ζ exert is a key mechanism in liver tumorigenesis.