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1.
BMJ Open Respir Res ; 11(1)2024 Oct 03.
Article in English | MEDLINE | ID: mdl-39362797

ABSTRACT

RATIONALE: In chronic obstructive pulmonary disease (COPD), accurately estimating lung function from electronic health record (EHR) data would be beneficial but requires addressing complexities in clinically obtained testing. This study compared analytic methods for estimating rate of forced expiratory volume in one second (FEV1) change from EHR data. METHODS: We estimated rate of FEV1 change in patients with COPD from a single centre who had ≥3 outpatient tests spanning at least 1 year. Estimates were calculated as both an absolute mL/year and a relative %/year using non-regressive (Total Change, Average Change) and regressive (Quantile, RANSAC, Huber) methods. We compared distributions of the estimates across methods focusing on extreme values. Univariate zero-inflated negative binomial regressions tested associations between estimates and all-cause or COPD hospitalisations. Results were validated in an external cohort. RESULTS: Among 1417 participants, median rate of change was approximately -30 mL/year or -2%/year. Non-regressive methods frequently generated erroneous estimates due to outlier first measurements or short intervals between tests. Average change yielded the most extreme estimates (minimum=-3761 mL/year), while regressive methods, and Huber specifically, minimised extreme estimates. Huber, Total Change and Quantile FEV1 slope estimates were associated with all-cause hospitalisations (Huber incidence rate ratio 0.98, 95% CI 0.97 to 0.99, p<0.001). Huber estimates were also associated with smoking status, comorbidities and prior hospitalisations. Similar results were identified in an external validation cohort. CONCLUSIONS: Using EHR data to estimate FEV1 rate of change is clinically applicable but sensitive to challenges intrinsic to clinically obtained data. While no analytic method will fully overcome these complexities, we identified Huber regression as useful in defining an individual's lung function change using EHR data.


Subject(s)
Electronic Health Records , Pulmonary Disease, Chronic Obstructive , Spirometry , Humans , Spirometry/methods , Forced Expiratory Volume , Pulmonary Disease, Chronic Obstructive/physiopathology , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Disease, Chronic Obstructive/epidemiology , Male , Female , Aged , Middle Aged , Lung/physiopathology , Hospitalization/statistics & numerical data
2.
Cell Chem Biol ; 30(11): 1453-1467.e8, 2023 11 16.
Article in English | MEDLINE | ID: mdl-37607550

ABSTRACT

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.


Subject(s)
Antineoplastic Agents , Colonic Neoplasms , Humans , DNA Mismatch Repair , Antineoplastic Agents/pharmacology , Mutagenesis , Cytotoxins
3.
bioRxiv ; 2023 Feb 21.
Article in English | MEDLINE | ID: mdl-36865268

ABSTRACT

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and in some cases, new therapeutic leads. In select cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

4.
iScience ; 24(12): 103440, 2021 Dec 17.
Article in English | MEDLINE | ID: mdl-34877497

ABSTRACT

Organoids mimic the physiologic and pathologic events of organs. However, no consensus on esophageal organoid (EO) culture methods has been reached. Moreover, organoid models reproducing esophageal squamous cell carcinoma (ESCC) initiation have been unavailable. Herein, we sought to develop an esophageal minimum essential organoid culture medium (E-MEOM) for culturing murine EOs and establishing an early ESCC model. We formulated E-MEOM to grow EOs from a single cell with clonal expansion, maintenance, and passage. We found that EOs cultured in E-MEOM were equivalent to the esophageal epithelium by histological analysis and transcriptomic study. Trp53 knockout and Kras G12D expression in EOs induced the development of esophageal squamous neoplasia, an early lesion of ESCC. Here we propose the new formula for EO culture with minimum components and the organoid model recapitulating ESCC initiation, laying the foundation for ESCC research and drug discovery.

5.
Mol Cell ; 81(8): 1698-1714.e6, 2021 04 15.
Article in English | MEDLINE | ID: mdl-33626321

ABSTRACT

The DREAM complex orchestrates cell quiescence and the cell cycle. However, how the DREAM complex is deregulated in cancer remains elusive. Here, we report that PAF (PCLAF/KIAA0101) drives cell quiescence exit to promote lung tumorigenesis by remodeling the DREAM complex. PAF is highly expressed in lung adenocarcinoma (LUAD) and is associated with poor prognosis. Importantly, Paf knockout markedly suppressed LUAD development in mouse models. PAF depletion induced LUAD cell quiescence and growth arrest. PAF is required for the global expression of cell-cycle genes controlled by the repressive DREAM complex. Mechanistically, PAF inhibits DREAM complex formation by binding to RBBP4, a core DREAM subunit, leading to transactivation of DREAM target genes. Furthermore, pharmacological mimicking of PAF-depleted transcriptomes inhibited LUAD tumor growth. Our results unveil how the PAF-remodeled DREAM complex bypasses cell quiescence to promote lung tumorigenesis and suggest that the PAF-DREAM axis may be a therapeutic vulnerability in lung cancer.


Subject(s)
Carcinogenesis/genetics , DNA-Binding Proteins/genetics , Kv Channel-Interacting Proteins/genetics , Lung Neoplasms/genetics , Lung/pathology , Repressor Proteins/genetics , A549 Cells , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Animals , Carcinogenesis/pathology , Cell Division/genetics , Cell Line , Cell Line, Tumor , Cell Proliferation/genetics , Female , Humans , Lung Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Nude , NIH 3T3 Cells , Transcriptional Activation/genetics , Transcriptome/genetics
6.
Hepatology ; 73(2): 776-794, 2021 02.
Article in English | MEDLINE | ID: mdl-32380568

ABSTRACT

BACKGROUND AND AIMS: How Wnt signaling is orchestrated in liver regeneration and tumorigenesis remains elusive. Recently, we identified transmembrane protein 9 (TMEM9) as a Wnt signaling amplifier. APPROACH AND RESULTS: TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation. TMEM9 is highly expressed in regenerating liver and hepatocellular carcinoma (HCC) cells. TMEM9 expression is enriched in the hepatocytes around the central vein and acutely induced by injury. In mice, Tmem9 knockout impairs hepatic regeneration with aberrantly increased adenomatosis polyposis coli (Apc) and reduced Wnt signaling. Mechanistically, TMEM9 down-regulates APC through lysosomal protein degradation through v-ATPase. In HCC, TMEM9 is overexpressed and necessary to maintain ß-catenin hyperactivation. TMEM9-up-regulated APC binds to and inhibits nuclear translocation of ß-catenin, independent of HCC-associated ß-catenin mutations. Pharmacological blockade of TMEM9-v-ATPase or lysosomal degradation suppresses Wnt/ß-catenin through APC stabilization and ß-catenin cytosolic retention. CONCLUSIONS: Our results reveal that TMEM9 hyperactivates Wnt signaling for liver regeneration and tumorigenesis through lysosomal degradation of APC.


Subject(s)
Adenomatous Polyposis Coli Protein/metabolism , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Membrane Proteins/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Adenomatous Polyposis Coli Protein/genetics , Animals , Carbon Tetrachloride/administration & dosage , Carbon Tetrachloride/toxicity , Carcinogenesis/pathology , Carcinoma, Hepatocellular/genetics , Cell Nucleus/metabolism , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/pathology , Disease Models, Animal , Gene Knockout Techniques , HEK293 Cells , Hep G2 Cells , Humans , Leupeptins/pharmacology , Liver Neoplasms/genetics , Liver Regeneration , Lysosomes/drug effects , Lysosomes/metabolism , Male , Membrane Proteins/genetics , Mice , Mice, Knockout , Proteolysis/drug effects , Wnt Signaling Pathway , Xenograft Model Antitumor Assays , beta Catenin/genetics , beta Catenin/metabolism
7.
Commun Biol ; 3(1): 257, 2020 05 22.
Article in English | MEDLINE | ID: mdl-32444826

ABSTRACT

Obesity is a global epidemic that is caused by excessive energy intake or inefficient energy expenditure. Brown or beige fat dissipates energy as heat through non-shivering thermogenesis by their high density of mitochondria. However, how the mitochondrial stress-induced signal is coupled to the cellular thermogenic program remains elusive. Here, we show that mitochondrial DNA escape-induced activation of the cGAS-STING pathway negatively regulates thermogenesis in fat-specific DsbA-L knockout mice, a model of adipose tissue mitochondrial stress. Conversely, fat-specific overexpression of DsbA-L or knockout of STING protects mice against high-fat diet-induced obesity. Mechanistically, activation of the cGAS-STING pathway in adipocytes activated phosphodiesterase PDE3B/PDE4, leading to decreased cAMP levels and PKA signaling, thus reduced thermogenesis. Our study demonstrates that mitochondrial stress-activated cGAS-STING pathway functions as a sentinel signal that suppresses thermogenesis in adipose tissue. Targeting adipose cGAS-STING pathway may thus be a potential therapeutic strategy to counteract overnutrition-induced obesity and its associated metabolic diseases.


Subject(s)
Glutathione Transferase/physiology , Membrane Proteins/metabolism , Mitochondria/pathology , Nucleotidyltransferases/metabolism , Obesity/etiology , Overnutrition/complications , Thermogenesis , Adipocytes/metabolism , Adipocytes/pathology , Animals , Diet, High-Fat , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/metabolism , Nucleotidyltransferases/genetics , Obesity/metabolism , Obesity/pathology , Stress, Physiological
8.
Proc Natl Acad Sci U S A ; 114(46): 12196-12201, 2017 11 14.
Article in English | MEDLINE | ID: mdl-29087318

ABSTRACT

Chronic inflammation in adipose tissue plays a key role in obesity-induced insulin resistance. However, the mechanisms underlying obesity-induced inflammation remain elusive. Here we show that obesity promotes mtDNA release into the cytosol, where it triggers inflammatory responses by activating the DNA-sensing cGAS-cGAMP-STING pathway. Fat-specific knockout of disulfide-bond A oxidoreductase-like protein (DsbA-L), a chaperone-like protein originally identified in the mitochondrial matrix, impaired mitochondrial function and promoted mtDNA release, leading to activation of the cGAS-cGAMP-STING pathway and inflammatory responses. Conversely, fat-specific overexpression of DsbA-L protected mice against high-fat diet-induced activation of the cGAS-cGAMP-STING pathway and inflammation. Taken together, we identify DsbA-L as a key molecule that maintains mitochondrial integrity. DsbA-L deficiency promotes inflammation and insulin resistance by activating the cGAS-cGAMP-STING pathway. Our study also reveals that, in addition to its well-characterized roles in innate immune surveillance, the cGAS-cGAMP-STING pathway plays an important role in mediating obesity-induced metabolic dysfunction.


Subject(s)
DNA, Mitochondrial/metabolism , Glutathione Transferase/genetics , Insulin Resistance , Membrane Proteins/genetics , Nucleotidyltransferases/genetics , Obesity/genetics , 3T3-L1 Cells , Adipocytes/metabolism , Adipocytes/pathology , Animals , Diet, High-Fat/adverse effects , Gene Expression Regulation , Glutathione Transferase/deficiency , Humans , Inflammation , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/metabolism , Mitochondria/pathology , Nucleotidyltransferases/metabolism , Obesity/etiology , Obesity/metabolism , Obesity/pathology , Primary Cell Culture , Protein Isoforms/genetics , Protein Isoforms/metabolism , Signal Transduction
9.
Protein Cell ; 8(6): 446-454, 2017 06.
Article in English | MEDLINE | ID: mdl-28220393

ABSTRACT

Obesity, which underlies various metabolic and cardiovascular diseases, is a growing public health challenge for which established therapies are inadequate. Given the current obesity epidemic, there is a pressing need for more novel therapeutic strategies that will help adult individuals to manage their weight. One promising therapeutic intervention for reducing obesity is to enhance energy expenditure. Investigations into human brown fat and the recently discovered beige/brite fat have galvanized intense research efforts during the past decade because of their pivotal roles in energy dissipation. In this review, we summarize the evolution of human brown adipose tissue (hBAT) research and discuss new in vivo methodologies for evaluating energy expenditure in patients. We highlight the differences between human and mouse BAT by integrating and comparing their cellular morphology, function, and gene expression profiles. Although great advances in hBAT biology have been achieved in the past decade, more cellular models are needed to acquire a better understanding of adipose-specific processes and molecular mechanisms. Thus, this review also describes the development of a human brown fat cell line, which could provide promising mechanistic insights into hBAT function, signal transduction, and development. Finally, we focus on the therapeutic potential and current limitations of hBAT as an anti-glycemic, anti-lipidemic, and weight loss-inducing 'metabolic panacea'.


Subject(s)
Adipose Tissue, Beige , Adipose Tissue, Brown , Obesity , Adipose Tissue, Beige/metabolism , Adipose Tissue, Beige/pathology , Adipose Tissue, Brown/metabolism , Adipose Tissue, Brown/pathology , Animals , Cell Line , Energy Metabolism , Humans , Mice , Obesity/metabolism , Obesity/pathology , Obesity/therapy
10.
Science ; 341(6144): 1236566, 2013 Jul 26.
Article in English | MEDLINE | ID: mdl-23888043

ABSTRACT

The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs life span in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin, as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals.


Subject(s)
Peptides/metabolism , Proteins/chemistry , Proteins/metabolism , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/chemistry , TOR Serine-Threonine Kinases/metabolism , Amino Acid Motifs , Amino Acids/metabolism , Animals , Cell Line , Culture Media , Humans , Mechanistic Target of Rapamycin Complex 1 , Mice , Multiprotein Complexes , Naphthyridines/pharmacology , Peptides/chemistry , Phosphorylation , Proteins/antagonists & inhibitors , TOR Serine-Threonine Kinases/antagonists & inhibitors
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