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Gastroenterology Sep 2023Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify...
Nucleolar HEAT Repeat Containing 1 Up-regulated by the Mechanistic Target of Rapamycin Complex 1 Signaling Promotes Hepatocellular Carcinoma Growth by Dominating Ribosome Biogenesis and Proteome Homeostasis.
BACKGROUND & AIMS
Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify critical ribosome biogenesis proteins that are overexpressed and crucial in HCC progression.
METHODS
HEAT repeat containing 1 (HEATR1) expression and clinical correlations were analyzed using The Cancer Genome Atlas and Gene Expression Omnibus databases and further evaluated by immunohistochemical analysis of an HCC tissue microarray. Gene expression was knocked down by small interfering RNA. HEATR1-knockdown cells were subjected to viability, cell cycle, and apoptosis assays and used to establish subcutaneous and orthotopic tumor models. Chromatin immunoprecipitation and quantitative polymerase chain reaction were performed to detect the association of candidate proteins with specific DNA sequences. Endogenous coimmunoprecipitation combined with mass spectrometry was used to identify protein interactions. We performed immunoblot and immunofluorescence assays to detect and localize proteins in cells. The nucleolus ultrastructure was detected by transmission electron microscopy. Click-iT (Thermo Fisher Scientific) RNA imaging and puromycin incorporation assays were used to measure nascent ribosomal RNA and protein synthesis, respectively. Proteasome activity, 20S proteasome foci formation, and protein stability were evaluated in HEATR1-knockdown HCC cells.
RESULTS
HEATR1 was the most up-regulated gene in a set of ribosome biogenesis mediators in HCC samples. High expression of HEATR1 was associated with poor survival and malignant clinicopathologic features in patients with HCC and contributed to HCC growth in vitro and in vivo. HEATR1 expression was regulated by the transcription factor specificity protein 1, which can be activated by insulin-like growth factor 1-mammalian target of rapamycin complex 1 signaling in HCC cells. HEATR1 localized predominantly in the nucleolus, bound to ribosomal DNA, and was associated with RNA polymerase I transcription/processing factors. Knockdown of HEATR1 disrupted ribosomal RNA biogenesis and impaired nascent protein synthesis, leading to reduced cytoplasmic proteasome activity and inhibitory-κB/nuclear factor-κB signaling. Moreover, HEATR1 knockdown induced nucleolar stress with increased nuclear proteasome activity and inactivation of the nucleophosmin 1-MYC axis.
CONCLUSIONS
Our study revealed that HEATR1 is up-regulated by insulin-like growth factor 1-mammalian target of rapamycin complex 1-specificity protein 1 signaling in HCC and functions as a crucial regulator of ribosome biogenesis and proteome homeostasis to promote HCC development.
Topics: Humans; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Homeostasis; Hot Temperature; Insulin-Like Growth Factor I; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Proteasome Endopeptidase Complex; Proteome; Ribosomes; RNA, Ribosomal
PubMed: 37247644
DOI: 10.1053/j.gastro.2023.05.029 -
Circulation Research Feb 2024The sympathoadrenergic system and its major effector PKA (protein kinase A) are activated to maintain cardiac output coping with physiological or pathological stressors....
BACKGROUND
The sympathoadrenergic system and its major effector PKA (protein kinase A) are activated to maintain cardiac output coping with physiological or pathological stressors. If and how PKA plays a role in physiological cardiac hypertrophy (PhCH) and pathological CH (PaCH) are not clear.
METHODS
Transgenic mouse models expressing the PKA inhibition domain (PKAi) of PKA inhibition peptide alpha (PKIalpha)-green fluorescence protein (GFP) fusion protein (PKAi-GFP) in a cardiac-specific and inducible manner (cPKAi) were used to determine the roles of PKA in physiological CH during postnatal growth or induced by swimming, and in PaCH induced by transaortic constriction (TAC) or augmented Ca influx. Kinase profiling was used to determine cPKAi specificity. Echocardiography was used to determine cardiac morphology and function. Western blotting and immunostaining were used to measure protein abundance and phosphorylation. Protein synthesis was assessed by puromycin incorporation and protein degradation by measuring protein ubiquitination and proteasome activity. Neonatal rat cardiomyocytes (NRCMs) infected with AdGFP (GFP adenovirus) or AdPKAi-GFP (PKAi-GFP adenovirus) were used to determine the effects and mechanisms of cPKAi on myocyte hypertrophy. rAAV9.PKAi-GFP was used to treat TAC mice.
RESULTS
(1) cPKAi delayed postnatal cardiac growth and blunted exercise-induced PhCH; (2) PKA was activated in hearts after TAC due to activated sympathoadrenergic system, the loss of endogenous PKIα (PKA inhibition peptide α), and the stimulation by noncanonical PKA activators; (3) cPKAi ameliorated PaCH induced by TAC and increased Ca influxes and blunted neonatal rat cardiomyocyte hypertrophy by isoproterenol and phenylephrine; (4) cPKAi prevented TAC-induced protein synthesis by inhibiting mTOR (mammalian target of rapamycin) signaling through reducing Akt (protein kinase B) activity, but enhancing inhibitory GSK-3α (glycogen synthase kinase-3α) and GSK-3β signals; (5) cPKAi reduced protein degradation by the ubiquitin-proteasome system via decreasing RPN6 phosphorylation; (6) cPKAi increased the expression of antihypertrophic atrial natriuretic peptide (ANP); (7) cPKAi ameliorated established PaCH and improved animal survival.
CONCLUSIONS
Cardiomyocyte PKA is a master regulator of PhCH and PaCH through regulating protein synthesis and degradation. cPKAi can be a novel approach to treat PaCH.
Topics: Mice; Rats; Animals; Proteasome Endopeptidase Complex; Cyclic AMP-Dependent Protein Kinases; Glycogen Synthase Kinase 3 beta; Cardiomegaly; Myocytes, Cardiac; Mice, Transgenic; Peptides; Mammals
PubMed: 38275112
DOI: 10.1161/CIRCRESAHA.123.322729 -
Pharmacological Research Aug 2023Hypoxia-inducible factor-2α (HIF-2α) is a transcription factor responsible for regulating genes related to angiogenesis and metabolism. This study aims to explore the...
Hypoxia-inducible factor-2α (HIF-2α) is a transcription factor responsible for regulating genes related to angiogenesis and metabolism. This study aims to explore the effect of a previously unreported mutation c.C2473T (p.R825S) in the C-terminal transactivation domain (CTAD) of HIF-2α that we detected in tissue of patients with liver disease. We sequenced available liver and matched blood samples obtained during partial liver resection or liver transplantation performed for clinical indications including hepatocellular carcinoma and liver failure. In tandem, we constructed cell lines and a transgenic mouse model bearing the corresponding identified mutation in HIF-2α from which we extracted primary hepatocytes. Lipid accumulation was evaluated in these cells and liver tissue from the mouse model using Oil Red O staining and biochemical measurements. We identified a mutation in the CTAD of HIF-2α (c.C2473T; p.R825S) in 5 of 356 liver samples obtained from patients with hepatopathy and dyslipidemia. We found that introduction of this mutation into the mouse model led to an elevated triglyceride level, lipid droplet accumulation in liver of the mutant mice and in their extracted primary hepatocytes, and increased transcription of genes related to hepatic fatty acid transport and synthesis in the mutant compared to the control groups. In mutant mice and cells, the protein levels of nuclear HIF-2α and its target perilipin-2 (PLIN2), a lipid droplet-related gene, were also elevated. Decreased lipophagy was observed in mutant groups. Our study defines a subpopulation of dyslipidemia that is caused by this HIF-2α mutation. This may have implications for personalized treatment.
Topics: Animals; Humans; Mice; Basic Helix-Loop-Helix Transcription Factors; Dyslipidemias; Lipids; Liver Neoplasms; Mutation
PubMed: 37453673
DOI: 10.1016/j.phrs.2023.106851 -
Clinical and Translational Medicine Nov 2023Translation dysregulation plays a crucial role in tumourigenesis and cancer progression. Oncogenic translation relies on the stability and availability of tRNAs for...
BACKGROUND
Translation dysregulation plays a crucial role in tumourigenesis and cancer progression. Oncogenic translation relies on the stability and availability of tRNAs for protein synthesis, making them potential targets for cancer therapy.
METHODS
This study performed immunohistochemistry analysis to assess NSUN2 levels in thyroid cancer. Furthermore, to elucidate the impact of NSUN2 on anaplastic thyroid cancer (ATC) malignancy, phenotypic assays were conducted. Drug inhibition and time-dependent plots were employed to analyse drug resistance. Liquid chromatography-mass spectrometry and bisulphite sequencing were used to investigate the m C methylation of tRNA at both global and single-base levels. Puromycin intake and high-frequency codon reporter assays verified the protein translation level. By combining mRNA and ribosome profiling, a series of downstream proteins and codon usage bias were identified. The acquired data were further validated by tRNA sequencing.
RESULTS
This study observed that the tRNA m C methyltransferase NSUN2 was up-regulated in ATC and is associated with dedifferentiation. Furthermore, NSUN2 knockdown repressed ATC formation, proliferation, invasion and migration both in vivo and in vitro. Moreover, NSUN2 repression enhanced the sensitivity of ATC to genotoxic drugs. Mechanically, NSUN2 catalyses tRNA structure-related m C modification, stabilising tRNA that maintains homeostasis and rapidly transports amino acids, particularly leucine. This stable tRNA has a substantially increased efficiency necessary to support a pro-cancer translation program including c-Myc, BCL2, RAB31, JUNB and TRAF2. Additionally, the NSUN2-mediated variations in m5C levels and different tRNA Leu iso-decoder families, partially contribute to a codon-dependent translation bias. Surprisingly, targeting NSUN2 disrupted the c-Myc to NSUN2 cycle in ATC.
CONCLUSIONS
This research revealed that a pro-tumour m5C methyltransferase, dynamic tRNA stability regulation and downstream oncogenes, c-Myc, elicits a codon-dependent oncogenic translation network that enhances ATC growth and formation. Furthermore, it provides new opportunities for targeting translation reprogramming in cancer cells.
Topics: Humans; Thyroid Carcinoma, Anaplastic; Methyltransferases; Codon; Thyroid Neoplasms; RNA, Transfer; rab GTP-Binding Proteins
PubMed: 37983928
DOI: 10.1002/ctm2.1466 -
Biochemistry and Cell Biology =... Dec 2023NDUFA4 is a component of respiratory chain-oxidative phosphorylation pathway. NDUFA4 is highly expressed in tumor tissues, but little is known about the function of...
NDUFA4 is a component of respiratory chain-oxidative phosphorylation pathway. NDUFA4 is highly expressed in tumor tissues, but little is known about the function of NDUFA4 in head and neck paraganglioma (HNPGL). We examined NDUFA4 expression in tissues from 10 HNPGL patients and 6 controls using qRT-PCR and Western blotting. NDUFA4 knockdown PGL-626 cells were established by using lentivirus infection and puromycin screening. Cell viability, ATP production, lipid reactive oxygen species, and mitochondrial membrane potential assays were performed to investigate the ferroptotic effects in NDUFA4 deficiency HNPGL cancer cells. Xenograft mouse model was created to detect the synergetic antitumor action between NDUFA4 deficiency and Metformin. NDUFA4 was upregulated in tumor tissues of HNPGL patients. NDUFA4 knockdown impaired the assembly of mitochondrial respiratory chain complexes and decreased the production of ATP and reduced cancer cell viability. Mechanistically, NDUFA4 knockdown increased cell ferroptosis, which further promoted Metformin-induced ferroptosis in PGL-626 cells. Therefore, NDUFA4 deficiency enhanced Metformin-mediated inhibition of the HNPGL progression in mice. In conclusion, NDUFA4 promotes the progression of HNPGL, and NDUFA4 knockdown enhances Metformin-mediated inhibition of the HNPGL progression in a mouse model.
Topics: Humans; Mice; Animals; Ferroptosis; Reactive Oxygen Species; Paraganglioma; Metformin; Adenosine Triphosphate; Electron Transport Complex IV
PubMed: 37602474
DOI: 10.1139/bcb-2023-0018 -
PloS One 2023Puromycin-sensitive aminopeptidase (E.C. 3.4.11.14, UniProt P55786), a zinc metallopeptidase belonging to the M1 family, degrades a number of bioactive peptides as well...
Puromycin-sensitive aminopeptidase (E.C. 3.4.11.14, UniProt P55786), a zinc metallopeptidase belonging to the M1 family, degrades a number of bioactive peptides as well as peptides released from the proteasome, including polyglutamine. We report the crystal structure of PSA at 2.3 Ǻ. Overall, the enzyme adopts a V-shaped architecture with four domains characteristic of the M1 family aminopeptidases, but it is in a less compact conformation compared to most M1 enzymes of known structure. A microtubule binding sequence is present in a C-terminal HEAT repeat domain of the enzyme in a position where it might serve to mediate interaction with tubulin. In the catalytic metallopeptidase domain, an elongated active site groove lined with aromatic and hydrophobic residues and a large S1 subsite may play a role in broad substrate recognition. The structure with bound polyglutamine shows a possible interacting mode of this peptide, which is supported by mutation.
Topics: Aminopeptidases; Peptides; Metalloproteases; Binding Sites; Substrate Specificity
PubMed: 37440518
DOI: 10.1371/journal.pone.0287086 -
Nephrology, Dialysis, Transplantation :... Nov 2023Glucocorticoids are the treatment of choice for proteinuric patients with minimal-change disease (MCD) and primary focal and segmental glomerulosclerosis (FSGS)....
BACKGROUND AND HYPOTHESIS
Glucocorticoids are the treatment of choice for proteinuric patients with minimal-change disease (MCD) and primary focal and segmental glomerulosclerosis (FSGS). Immunosuppressive as well as direct effects on podocytes are believed to mediate their actions. In this study, we analyzed the anti-proteinuric effects of inhibition of the glucocorticoid receptor (GR) in glomerular epithelial cells, including podocytes.
METHODS
We employed genetic and pharmacological approaches to inhibit the GR. Genetically, we used Pax8-Cre/GRfl/fl mice to specifically inactivate the GR in kidney epithelial cells. Pharmacologically, we utilized a glucocorticoid antagonist called mifepristone.
RESULTS
Genetic inactivation of GR, specifically in kidney epithelial cells, using Pax8-Cre/GRfl/fl mice, ameliorated proteinuria following protein overload. We further tested the effects of pharmacological GR inhibition in three models and species: the puromycin-aminonucleoside-induced nephrosis model in rats, the protein overload model in mice and the inducible transgenic NTR/MTZ zebrafish larvae with specific and reversible podocyte injury. In all three models, both pharmacological GR activation and inhibition consistently and significantly ameliorated proteinuria. Additionally, we translated our findings to humans, where three nephrotic adult patients with MCD or primary FSGS with contraindications or insufficient responses to corticosteroids, were treated with mifepristone. This treatment resulted in a clinically relevant reduction of proteinuria.
CONCLUSIONS
Thus, across multiple species and proteinuria models, both genetic and pharmacological GR inhibition was at least as effective as pronounced GR activation. While, the mechanism remains perplexing, GR inhibition may be a novel and targeted therapeutic approach to treat glomerular proteinuria potentially bypassing adverse actions of steroids.
PubMed: 38037533
DOI: 10.1093/ndt/gfad254 -
Small Methods Jul 2023Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the...
Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the targeted gene product. While these approaches are rather straightforward for membrane-bound proteins, they can be laborious for intracellular proteins, given that selection of full knockout (KO) cells often requires the amplification of single-cell clones. Moreover, viral-mediated delivery systems, besides the Cas9 and gRNA, lead to the integration of unwanted genetic material, such as antibiotic resistance genes, introducing experimental biases. Here, an alternative non-viral delivery approach is presented for CRISPR/Cas9, allowing efficient and flexible selection of KO polyclonal cells. This all-in-one mammalian CRISPR-Cas9 expression vector, ptARgenOM, encodes the gRNA and the Cas9 linked to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein and the puromycin N-acetyltransferase, allowing for transient, expression-dependent selection and enrichment of isogenic KO cells. After evaluation using more than 12 distinct targets in 6 cell lines, ptARgenOM is found to be efficient in producing KO cells, reducing the time required to obtain a polyclonal isogenic cell line by 4-6 folds. Altogether ptARgenOM provides a simple, fast, and cost-effective delivery tool for genome editing.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Cell Line; Mammals
PubMed: 37156748
DOI: 10.1002/smtd.202300069 -
Cold Spring Harbor Protocols Jun 2024The selection of mosquito transgenic larvae using a nonfluorescent approach can be advantageous to reserve fluorophores for downstream applications, such as...
The selection of mosquito transgenic larvae using a nonfluorescent approach can be advantageous to reserve fluorophores for downstream applications, such as immunostaining or for the study of promoter activity by cloning a fluorescence reporter gene under the control of that promoter. We previously reported that puromycin selection is efficient in transgenic or larvae expressing an selection marker. A concentration of puromycin of >10 µg/mL is lethal for larvae, unless they carry the resistance gene, conferring them resistance to puromycin concentrations of 25-80 µg/mL. A drawback of this fully dominant selection marker is that, unlike with fluorescence markers, homozygous transgenics cannot be distinguished from heterozygotes. Here, we outline the procedure for selecting puromycin-resistant transgenic larvae.
Topics: Animals; Puromycin; Larva; Animals, Genetically Modified; Anopheles; Selection, Genetic
PubMed: 37696571
DOI: 10.1101/pdb.prot108308 -
Journal of Experimental & Clinical... Dec 2023Colorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism. Ferroptosis, a programmed cell death dependent on iron,...
BACKGROUND
Colorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism. Ferroptosis, a programmed cell death dependent on iron, has emerged as a promising strategy for CRC treatment. Although small nucleolar RNAs are extensively involved in carcinogenesis, it is unclear if they regulate ferroptosis during CRC pathogenesis.
METHODS
The dysregulated snoRNAs were identified using published sequencing data of CRC tissues. The expression of the candidate snoRNAs, host gene and target gene were assessed by real-time quantitative PCR (RT-qPCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and western blots. The biological function of critical molecules was investigated using in vitro and in vivo strategies including Cell Counting Kit-8 (CCK8), colony formation assay, flow cytometry, Fe/Fe, GSH/GSSG and the xenograft mice models. The ribosomal activities were determined by polysome profiling and O-propargyl-puromycin (OP-Puro) assay. The proteomics was conducted to clarify the downstream targets and the underlying mechanisms were validated by IHC, Pearson correlation analysis, protein stability and rescue assays. The clinical significance of the snoRNA was explored using the Cox proportional hazard model, receiver operating characteristic (ROC) and survival analysis.
RESULTS
Here, we investigated the SNORA56, which was elevated in CRC tissues and plasma, and correlated with CRC prognosis. SNORA56 deficiency in CRC impaired proliferation and triggered ferroptosis, resulting in reduced tumorigenesis. Mechanistically, SNORA56 mediated the pseudouridylation of 28 S rRNA at the U1664 site and promoted the translation of the catalytic subunit of glutamate cysteine ligase (GCLC), an indispensable rate-limiting enzyme in the biosynthesis of glutathione, which can inhibit ferroptosis by suppressing lipid peroxidation.
CONCLUSIONS
Therefore, the SNORA56/28S rRNA/GCLC axis stimulates CRC progression by inhibiting the accumulation of cellular peroxides, and it may provide biomarker and therapeutic applications in CRC.
Topics: Animals; Humans; Mice; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Ferroptosis; Glutamate-Cysteine Ligase; In Situ Hybridization, Fluorescence; RNA, Ribosomal; RNA, Small Nuclear
PubMed: 38049865
DOI: 10.1186/s13046-023-02906-8