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1.
Mol Ther ; 26(8): 1983-1995, 2018 08 01.
Article in English | MEDLINE | ID: mdl-29914758

ABSTRACT

Primary hyperoxalurias (PHs) are autosomal recessive disorders caused by the overproduction of oxalate leading to calcium oxalate precipitation in the kidney and eventually to end-stage renal disease. One promising strategy to treat PHs is to reduce the hepatic production of oxalate through substrate reduction therapy by inhibiting liver-specific glycolate oxidase (GO), which controls the conversion of glycolate to glyoxylate, the proposed main precursor to oxalate. Alternatively, diminishing the amount of hepatic lactate dehydrogenase (LDH) expression, the proposed key enzyme responsible for converting glyoxylate to oxalate, should directly prevent the accumulation of oxalate in PH patients. Using RNAi, we provide the first in vivo evidence in mammals to support LDH as the key enzyme responsible for converting glyoxylate to oxalate. In addition, we demonstrate that reduction of hepatic LDH achieves efficient oxalate reduction and prevents calcium oxalate crystal deposition in genetically engineered mouse models of PH types 1 (PH1) and 2 (PH2), as well as in chemically induced PH mouse models. Repression of hepatic LDH in mice did not cause any acute elevation of circulating liver enzymes, lactate acidosis, or exertional myopathy, suggesting further evaluation of liver-specific inhibition of LDH as a potential approach for treating PH1 and PH2 is warranted.


Subject(s)
Hyperoxaluria, Primary/therapy , L-Lactate Dehydrogenase/antagonists & inhibitors , Oxalates/metabolism , RNA Interference/physiology , Animals , Disease Models, Animal , Gene Silencing , Humans , Hyperoxaluria, Primary/genetics , Hyperoxaluria, Primary/metabolism , L-Lactate Dehydrogenase/genetics , Liver/enzymology , Mice
2.
Mol Ther ; 26(7): 1771-1782, 2018 07 05.
Article in English | MEDLINE | ID: mdl-29784585

ABSTRACT

Glycogen storage diseases (GSDs) of the liver are devastating disorders presenting with fasting hypoglycemia as well as hepatic glycogen and lipid accumulation, which could lead to long-term liver damage. Diet control is frequently utilized to manage the potentially dangerous hypoglycemia, but there is currently no effective pharmacological treatment for preventing hepatomegaly and concurrent liver metabolic abnormalities, which could lead to fibrosis, cirrhosis, and hepatocellular adenoma or carcinoma. In this study, we demonstrate that inhibition of glycogen synthesis using an RNAi approach to silence hepatic Gys2 expression effectively prevents glycogen synthesis, glycogen accumulation, hepatomegaly, fibrosis, and nodule development in a mouse model of GSD III. Mechanistically, reduction of accumulated abnormally structured glycogen prevents proliferation of hepatocytes and activation of myofibroblasts as well as infiltration of mononuclear cells. Additionally, we show that silencing Gys2 expression reduces hepatic steatosis in a mouse model of GSD type Ia, where we hypothesize that the reduction of glycogen also reduces the production of excess glucose-6-phosphate and its subsequent diversion to lipid synthesis. Our results support therapeutic silencing of GYS2 expression to prevent glycogen and lipid accumulation, which mediate initial signals that subsequently trigger cascades of long-term liver injury in GSDs.


Subject(s)
Glycogen Storage Disease Type III/genetics , Glycogen Synthase/genetics , Glycogen/genetics , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Liver/pathology , RNA Interference/physiology , Animals , Disease Models, Animal , Female , Fibroblasts/pathology , Glucose-6-Phosphate/genetics , Glycogen Storage Disease Type III/pathology , Hepatocytes/pathology , Hepatomegaly/genetics , Male , Mice , Mice, Inbred C57BL
3.
Mol Ther ; 24(4): 770-8, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26758691

ABSTRACT

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, metabolic disorder caused by mutations of alanine-glyoxylate aminotransferase (AGT), a key hepatic enzyme in the detoxification of glyoxylate arising from multiple normal metabolic pathways to glycine. Accumulation of glyoxylate, a precursor of oxalate, leads to the overproduction of oxalate in the liver, which accumulates to high levels in kidneys and urine. Crystalization of calcium oxalate (CaOx) in the kidney ultimately results in renal failure. Currently, the only treatment effective in reduction of oxalate production in patients who do not respond to high-dose vitamin B6 therapy is a combined liver/kidney transplant. We explored an alternative approach to prevent glyoxylate production using Dicer-substrate small interfering RNAs (DsiRNAs) targeting hydroxyacid oxidase 1 (HAO1) mRNA which encodes glycolate oxidase (GO), to reduce the hepatic conversion of glycolate to glyoxylate. This approach efficiently reduces GO mRNA and protein in the livers of mice and nonhuman primates. Reduction of hepatic GO leads to normalization of urine oxalate levels and reduces CaOx deposition in a preclinical mouse model of PH1. Our results support the use of DsiRNA to reduce liver GO levels as a potential therapeutic approach to treat PH1.


Subject(s)
Alcohol Oxidoreductases/genetics , Calcium Oxalate/metabolism , Hyperoxaluria, Primary/therapy , RNA, Small Interfering/administration & dosage , Animals , DEAD-box RNA Helicases/metabolism , Disease Models, Animal , Glyoxylates/urine , Humans , Hyperoxaluria, Primary/enzymology , Hyperoxaluria, Primary/urine , Liver/metabolism , Mice , Nanoparticles/chemistry , RNA, Small Interfering/pharmacology , Ribonuclease III/metabolism
4.
J Biol Chem ; 285(41): 31139-47, 2010 Oct 08.
Article in English | MEDLINE | ID: mdl-20682773

ABSTRACT

Transcription factor LSF is essential for cell cycle progression, being required for activating expression of the thymidylate synthase (Tyms) gene at the G1/S transition. We previously established that phosphorylation of LSF in early G1 at Ser-291 and Ser-309 inhibits its transcriptional activity and that dephosphorylation later in G1 is required for its reactivation. Here we reveal the role of prolyl cis-trans isomerase Pin1 in activating LSF, by facilitating dephosphorylation at both Ser-291 and Ser-309. We demonstrate that Pin1 binds LSF both in vitro and in vivo. Using coimmunoprecipitation assays, we identify three SP/TP motifs in LSF (at residues Ser-291, Ser-309, and Thr-329) that are required and sufficient for association with Pin1. Co-expression of Pin1 enhances LSF transactivation potential in reporter assays. The Pin1-dependent enhancement of LSF activity requires residue Thr-329 in LSF, requires both the WW and PPiase domains of Pin1, and correlates with hypophosphorylation of LSF at Ser-291 and Ser-309. These findings support a model in which the binding of Pin1 at the Thr-329-Pro-330 motif in LSF permits isomerization by Pin1 of the peptide bonds at the nearby phosphorylated SP motifs (Ser-291 and Ser-309) to the trans configuration, thereby facilitating their dephosphorylation.


Subject(s)
DNA-Binding Proteins/metabolism , Peptidylprolyl Isomerase/metabolism , Transcription Factors/metabolism , Amino Acid Motifs , Animals , DNA-Binding Proteins/genetics , Mice , NIH 3T3 Cells , NIMA-Interacting Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/genetics , Phosphorylation/physiology , Protein Structure, Tertiary , Transcription Factors/genetics
5.
Proc Natl Acad Sci U S A ; 107(18): 8357-62, 2010 May 04.
Article in English | MEDLINE | ID: mdl-20404171

ABSTRACT

Hepatocellular carcinoma (HCC) is a highly aggressive cancer with no currently available effective treatment. Understanding of the molecular mechanism of HCC development and progression is imperative for developing novel, effective, and targeted therapies for this lethal disease. In this article, we document that the cellular transcription factor Late SV40 Factor (LSF) plays an important role in HCC pathogenesis. LSF protein was significantly overexpressed in human HCC cells compared to normal hepatocytes. In 109 HCC patients, LSF protein was overexpressed in >90% cases, compared to normal liver, and LSF expression level showed significant correlation with the stages and grades of the disease. Forced overexpression of LSF in less aggressive HCC cells resulted in highly aggressive, angiogenic, and multiorgan metastatic tumors in nude mice. Conversely, inhibition of LSF significantly abrogated growth and metastasis of highly aggressive HCC cells in nude mice. Microarray studies revealed that as a transcription factor, LSF modulated specific genes regulating invasion, angiogenesis, chemoresistance, and senescence. The expression of osteopontin (OPN), a gene regulating every step in tumor progression and metastasis, was robustly up-regulated by LSF. It was documented that LSF transcriptionally up-regulates OPN, and loss-of-function studies demonstrated that OPN plays an important role in mediating the oncogenic functions of LSF. Together, these data establish a regulatory role of LSF in cancer, particularly HCC pathogenesis, and validate LSF as a viable target for therapeutic intervention.


Subject(s)
Carcinoma, Hepatocellular/metabolism , DNA-Binding Proteins/metabolism , Liver Neoplasms/metabolism , Oncogenes , Transcription Factors/metabolism , Animals , Carcinoma, Hepatocellular/blood supply , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Proliferation , Cells, Cultured , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/blood supply , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice , Mice, Nude , Neoplasm Metastasis , Neoplasm Transplantation , Osteopontin/genetics , Osteopontin/metabolism , RNA Interference , Rats , Tissue Array Analysis , Transcription Factors/genetics , Transcription, Genetic , Up-Regulation
6.
Proc Natl Acad Sci U S A ; 106(31): 12938-43, 2009 Aug 04.
Article in English | MEDLINE | ID: mdl-19622726

ABSTRACT

Astrocyte elevated gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) patients and plays a significant role in mediating aggressive progression of HCC. AEG-1 is known to augment invasion, metastasis, and angiogenesis, and we now demonstrate that AEG-1 directly contributes to another important hallmark of aggressive cancers, that is, resistance to chemotherapeutic drugs, such as 5-fluorouracil (5-FU). AEG-1 augments expression of the transcription factor LSF that regulates the expression of thymidylate synthase (TS), a target of 5-FU. In addition, AEG-1 enhances the expression of dihydropyrimidine dehydrogenase (DPYD) that catalyzes the initial and rate-limiting step in the catabolism of 5-FU. siRNA-mediated inhibition of AEG-1, LSF, or DPYD significantly increased the sensitivity of HCC cells to 5-FU in vitro and a lentivirus delivering AEG-1 siRNA in combination with 5-FU markedly inhibited growth of HCC cells xenotransplanted in athymic nude mice when compared to either agent alone. The present studies highlight 2 previously unidentified genes, AEG-1 and LSF, contributing to chemoresistance. Inhibition of AEG-1 might be exploited as a therapeutic strategy along with 5-FU-based combinatorial chemotherapy for HCC, a highly fatal cancer with currently very limited therapeutic options.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Carcinoma, Hepatocellular/drug therapy , Cell Adhesion Molecules/genetics , DNA-Binding Proteins/genetics , Fluorouracil/pharmacology , Liver Neoplasms/drug therapy , Transcription Factors/genetics , Animals , Carcinoma, Hepatocellular/genetics , Cell Adhesion Molecules/antagonists & inhibitors , Cell Line, Tumor , DNA/metabolism , DNA-Binding Proteins/physiology , Dihydrouracil Dehydrogenase (NADP)/physiology , Drug Resistance, Neoplasm/genetics , Humans , Ki-67 Antigen/analysis , Liver Neoplasms/genetics , Membrane Proteins , Mice , RNA-Binding Proteins , Thymidylate Synthase/genetics , Transcription Factors/physiology
7.
Cell Cycle ; 8(14): 2146-51, 2009 Jul 15.
Article in English | MEDLINE | ID: mdl-19556876

ABSTRACT

Cell cycle progression in mammalian cells from G(1) into S phase requires sensing and integration of multiple inputs, in order to determine whether to continue to cellular DNA replication and subsequently, to cell division. Passage to S requires transition through the restriction point, which at a molecular level consists of a bistable switch involving E2Fs and pRb family members. At the G(1)/S boundary, a number of genes essential for DNA replication and cell cycle progression are upregulated, promoting entry into S phase. Although the activating E2Fs are the most extensively characterized transcription factors driving G(1)/S expression, LSF is also a transcription factor essential for stimulating G(1)/S gene expression. A critical LSF target gene at this stage, Tyms, encodes thymidylate synthetase. In investigating how LSF is activated in a cell cycle-dependent manner, we recently identified a novel time delay mechanism for regulating its activity during G(1) progression, which is apparently independent of the E2F/pRb axis. This involves inhibition of LSF in early G(1) by two major proliferative signaling pathways: ERK and cyclin C/CDK, followed by gradual dephosphorylation during mid- to late-G(1). Whether LSF and E2F act independently or in concert to promote G(1)/S progression remains to be determined.


Subject(s)
DNA-Binding Proteins/metabolism , E2F Transcription Factors/metabolism , Resting Phase, Cell Cycle , S Phase , Transcription Factors/metabolism , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , DNA-Binding Proteins/chemistry , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Phosphorylation , Signal Transduction , Transcription Factors/chemistry
8.
Mol Cell Biol ; 29(9): 2335-45, 2009 May.
Article in English | MEDLINE | ID: mdl-19237534

ABSTRACT

Transcription factor LSF is required for progression from quiescence through the cell cycle, regulating thymidylate synthase (Tyms) expression at the G(1)/S boundary. Given the constant level of LSF protein from G(0) through S, we investigated whether LSF is regulated by phosphorylation in G(1). In vitro, LSF is phosphorylated by cyclin E/cyclin-dependent kinase 2 (CDK2), cyclin C/CDK2, and cyclin C/CDK3, predominantly on S309. Phosphorylation of LSF on S309 is maximal 1 to 2 h after mitogenic stimulation of quiescent mouse fibroblasts. This phosphorylation is mediated by cyclin C-dependent kinases, as shown by coimmunoprecipitation of LSF and cyclin C in early G(1) and by abrogation of LSF S309 phosphorylation upon suppression of cyclin C with short interfering RNA. Although mouse fibroblasts lack functional CDK3 (the partner of cyclin C in early G(1) in human cells), CDK2 compensates for this absence. By transient transfection assays, phosphorylation at S309, mediated by cyclin C overexpression, inhibits LSF transactivation. Moreover, overexpression of cyclin C and CDK3 inhibits induction of endogenous Tyms expression at the G(1)/S transition. These results identify LSF as only the second known target (in addition to pRb) of cyclin C/CDK activity during progression from quiescence to early G(1). Unexpectedly, this phosphorylation prevents induction of LSF target genes until late G(1).


Subject(s)
Cyclin-Dependent Kinase 2/metabolism , Cyclins/metabolism , DNA-Binding Proteins/metabolism , Fibroblasts/physiology , G1 Phase/physiology , Transcription Factors/metabolism , Transcription, Genetic , Animals , Cyclin C , Cyclin E/genetics , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 3 , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Cyclins/genetics , DNA-Binding Proteins/genetics , Enzyme Induction , Fibroblasts/cytology , Humans , Mice , NIH 3T3 Cells , Phosphorylation , Serine/metabolism , Thymidylate Synthase/genetics , Thymidylate Synthase/metabolism , Transcription Factors/genetics
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