Myotonic dystrophy type 1 (DM1) is caused by a CTG-repeat expansion in the DMPK gene. Expression of pathogenic expanded CUG-repeat (CUGexp) RNA causes multisystemic disease by perturbing the functions of RNA binding proteins, resulting in expression of fetal protein isoforms in adult tissues. Cardiac involvement affects 50% of individuals with DM1 and causes 25% of disease-related deaths. We developed a transgenic mouse model for tetracycline-inducible and heart-specific expression of human DMPK mRNA containing 960 CUG repeats. CUGexp RNA is expressed in atria and ventricles and induced mice exhibit electrophysiological and molecular features of DM1 disease including cardiac conduction delays, supraventricular arrhythmias, nuclear RNA foci with Muscleblind protein colocalization and alternative splicing defects. Importantly, these phenotypes were rescued upon loss of CUGexp RNA expression. Transcriptome analysis revealed gene expression and alternative splicing changes in ion transport genes that are associated with inherited cardiac conduction diseases, including a subset of genes involved in calcium handling. Consistent with RNA-seq results, calcium handling defects were identified in atrial cardiomyocytes isolated from mice expressing CUGexp RNA. These results identify potential tissue-specific mechanisms contributing to cardiac pathogenesis in DM1 and demonstrate the utility of reversible phenotypes in our model to facilitate development of targeted therapeutic approaches.
Ashish N. Rao, Hannah M. Campbell, Xiangnan Guan, Tarah A. Word, Xander H.T. Wehrens, Zheng Xia, Thomas A. Cooper
Triple negative breast cancers (TNBC) lack effective targeted therapies and cytotoxic chemotherapies remain the standard of care for this subtype. Owing to their increased genomic instability, PARP inhibitors (PARPi) are being tested against TNBCs. In particular, clinical trials are now interrogating the efficacy of PARPi combined with chemotherapies. Intriguingly, while response rates are low, cohorts of patients do respond. Moreover, recent studies suggest that an increase in levels of reactive oxygen species (ROS) may sensitize cells to PARPi. This represents a therapeutic opportunity, as several chemotherapies, including doxorubicin, function in part by producing ROS. We previously demonstrated that the p66ShcA adaptor protein is variably expressed in TNBCs. We now show that in response to therapy-induced stress, p66ShcA stimulates ROS production, which, in turn, potentiates synergy between doxorubicin/PARPi combination therapy in TNBCs. This p66ShcA-induced sensitivity relies on the accumulation of oxidative damage in TNBCs, rather than genomic instability, to potentiate cell death. These findings suggest that increasing the expression of p66ShcA protein levels in TNBCs represents a rational approach to bolster the synergy between PARPi and doxorubicin.
Eduardo Cepeda Cañedo, Stephanie Totten, Ryuhjin Ahn, Paul Savage, Deanna MacNeil, Jesse Hudson, Chantal Autexier, Genevieve Deblois, Morag Park, Michael Witcher, Josie Ursini-Siegel
Rewiring tumor cells to undergo drug-induced apoptosis could be a promising way to overcome chemoresistance, therefore identifying causative factors for chemoresistance is of high importance. Global proteome-profiling of sensitive, early and late cisplatin resistant OSCC lines identified CMTM6 as a top ranked up-regulated protein. Analyses of OSCC patient tumor samples demonstrated significantly higher CMTM6 expression in chemotherapy-non-responders as compared to responders. In addition, a significant association between higher CMTM6 expression and poorer relapse-free survival in ESCC, HNSCC was monitored from Kaplan-Meier-Plot analysis. Stable knockdown of CMTM6 restores cisplatin-mediated cell death in chemoresistant OSCC cell lines. Similarly, upon CMTM6 overexpression in CMTM6KD lines, the cisplatin resistant phenotype was efficiently rescued. The patient-derived cell xenograft model of chemoresistant OSCC displayed CMTM6 depletion restored the cisplatin-induced cell death and tumor burden significantly. The transcriptome analysis of CMTM6KD and control chemoresistant cells depicted enrichment of Wnt-signaling pathway. Mechanistically, we demonstrated that CMTM6 interaction with membrane bound Enolase-1 stabilized its expression, leading to AKT-GSK3β mediated activation of Wnt-signaling. CMTM6 has been identified as a stabilizer of PD-L1 thereby facilitates immune evasion by tumor cells. As CMTM6 facilitates tumor cells for immune evasion and mediates cisplatin resistance, it can be an important therapeutic target for therapy resistant OSCC.
Pallavi Mohapatra, Omprakash Shriwas, Sibasish Mohanty, Arup Ghosh, Shuchi Smita, Sandeep Rai Kaushik, Rakesh Arya, Rachna Rath, Saroj Das Majumdar, Dillip Kumar Muduly, Sunil Raghav, Ranjan K. Nanda, Rupesh Dash
Background: Loss-of-function variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are linked to human diseases with high risk of sudden death, including epileptic encephalopathy and cardiac arrhythmia. β1 subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming a subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Methods: We investigated regulated intramembrane proteolysis (RIP) of β1 by BACE1 and γ-secretase.Results: We show that β1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA-seq, we identified a subset of genes that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b null cardiac tissue which, by definition, lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo. Conclusion: We propose that human disease variants resulting in SCN1B loss-of-function cause transcriptional dysregulation that contributes to altered excitability. These results provide important new insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multi-functionality of sodium channel β1 subunits.
Alexandra A. Bouza, Nnamdi Edokobi, Samantha L. Hodges, Alexa M. Pinsky, James Offord, Lin Piao, Yan-Ting Zhao, Anatoli N. Lopatin, Luis F. Lopez-Santiago, Lori L. Isom
Osteosarcoma (OS) is an aggressive mesenchymal tumor for which no molecularly targeted therapies are available. We have previously identified TRAF2 and NCK-interacting protein kinase (TNIK) as an essential factor for the transactivation of Wnt signal target genes and shown that its inhibition leads to eradication of colorectal cancer stem cells. The involvement of Wnt signaling in the pathogenesis of OS has been implicated. The aim of the present study was to examine the potential of TNIK as a therapeutic target in OS. RNA interference or pharmacological inhibition of TNIK suppressed the proliferation of OS cells. Transcriptome analysis suggested that a small-molecule inhibitor of TNIK up-regulated the expression of genes involved in OS cell metabolism and down-regulated transcription factors essential for maintaining the stem cell phenotype. Metabolome analysis revealed that this TNIK inhibitor redirected the metabolic network from carbon flux towards lipid accumulation in OS cells. Using in vitro and in vivo OS models, we confirmed that TNIK inhibition abrogated the OS stem cell phenotype, simultaneously driving conversion of OS cells to adipocyte-like cells through induction of peroxisome proliferator-activated receptor-γ. In relation to potential therapeutic targeting in clinical practice, TNIK was confirmed to be in an active state in OS cell lines and clinical specimens. From these findings, we conclude that TNIK is applicable as a potential target for treatment of OS, affecting cell fate determination.
Toru Hirozane, Mari Masuda, Teppei Sugano, Tetsuya Sekita, Naoko Goto, Toru Aoyama, Takato Sakagami, Yuko Uno, Hideki Moriyama, Masaaki Sawa, Naofumi Asano, Masaya Nakamura, Morio Matsumoto, Robert Nakayama, Tadashi Kondo, Akira Kawai, Eisuke Kobayashi, Tesshi Yamada
Asymmetric cell division (ACD) enables the maintenance of a stem cell population while simultaneously generating differentiated progeny. Cancer stem cells (CSCs) undergo multiple modes of cell division during tumor expansion and in response to therapy, yet the functional consequences of these division modes remain to be determined. Using a fluorescent reporter for cell surface receptor distribution during mitosis, we found that ACD generated a daughter cell with enhanced therapeutic resistance and increased co-enrichment of epidermal growth factor receptor (EGFR) and neurotrophin receptor (p75NTR) from a glioblastoma CSC. Stimulation of both receptors antagonized differentiation induction and promoted self-renewal capacity. p75NTR knockdown enhanced the therapeutic efficacy of EGFR inhibition, indicating that co-inheritance of p75NTR and EGFR promotes resistance to EGFR inhibition through a redundant mechanism. These data demonstrate that ACD produces progeny with co-enriched growth factor receptors, which contributes to the generation of a more therapeutically resistant CSC population.
Masahiro Hitomi, Anastasia P. Chumakova, Daniel J. Silver, Arnon M. Knudsen, W. Dean Pontius, Stephanie Murphy, Neha S. Anand, Bjarne Winther Kristensen, Justin Lathia
COPD is a chronic respiratory disease characterized by small airway remodeling and alveolar emphysema due to environmental stresses such as cigarette smoking (CS). Oxidative stress is commonly implicated in COPD pathology, but recent findings suggest that one oxidant-producing NADPH oxidase homolog, dual oxidase 1 (DUOX1), is downregulated in the airways of COPD patients. We evaluated lung tissue sections from COPD patients for small airway epithelial DUOX1 protein expression, in association with measures of lung function and small airway and alveolar remodeling. We also addressed the impact of DUOX1 for lung tissue remodeling in mouse models of COPD. Small airway DUOX1 levels were decreased in advanced COPD, and correlated with loss of lung function and markers of emphysema and remodeling. Similarly, DUOX1 downregulation in correlation with extracellular matrix remodeling was observed in a genetic model of COPD, transgenic SPC-TNF-α mice. Finally, development of subepithelial airway fibrosis in mice due to exposure to the CS-component acrolein, or alveolar emphysema induced by administration of elastase, were in both cases exacerbated in Duox1-deficient mice. Collectively, our studies highlight that downregulation of DUOX1 may be a contributing feature of COPD pathogenesis, likely related to impaired DUOX1-mediated innate injury responses involved in epithelial homeostasis.
Caspar Schiffers, Cheryl van de Wetering, Robert A. Bauer, Aida Habibovic, Milena Hristova, Christopher M. Dustin, Sara Lambrichts, Pamela M. Vacek, Emiel F.M. Wouters, Niki L. Reynaert, Albert van der Vliet
Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short life spans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of ~ 40 days and died of gastrointestinal disease, but therapeutic regimens aimed at restoring gastrointestinal transit extended median survival to ~ 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression pattern in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the upper and lower airways. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1 year-old CF rabbits as compared to WT littermates, which were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.
Jie Xu, Alessandra Livraghi-Butrico, Xia Hou, Carthic Rajagopalan, Jifeng Zhang, Jun Song, Hong Jiang, Hong-guang Wei, Hui Wang, Mohamad Bouhamdan, Jinxue Ruan, Dongshan Yang, Yining Qiu, Xie Youming, Ronald P. Barrett, Sharon A. McClellan, Hongmei Mou, Qingtian Wu, Xuequn Chen, Troy D. Rogers, Kristen J. Wilkinson, Rodney C. Gilmore, Charles R. Esther Jr., Khalequz Zaman, Xiubin Liang, Michael Sobolic, Linda Hazlett, Kezhong Zhang, Raymond A. Frizzell, Martina Gentzsch, Wanda K. O'Neal, Barbara R. Grubb, Y Eugene Chen, Richard C. Boucher, Fei Sun
Evidence for reduced expression of cyclin G associated kinase (GAK) in glomeruli of patients with chronic kidney disease was observed in the Nephroseq human database, and GAK was found to be associated with the decline in kidney function. To examine the role of GAK, a protein that functions to uncoat clathrin during endocytosis, we generated podocyte-specific Gak-knockout mice (Gak-KO), which developed progressive proteinuria and kidney failure with global glomerulosclerosis. We isolated glomeruli from the mice carrying the mutation to perform messenger RNA profiling and unearthed evidence for dysregulated podocyte calpain protease activity as an important contributor to progressive podocyte damage. Treatment with calpain inhibitor III specifically inhibited calpain-1/-2 activities, mitigated the degree of proteinuria and glomerulosclerosis, and led to a striking increase in survival in the Gak-KO mice. Podocyte-specific deletion of Capns1, essential for calpain-1 and calpain-2 activities, also improved proteinuria and glomerulosclerosis in Gak-KO mice. Increased podocyte calpain activity–mediated proteolysis of IκBα resulted in increased NF-κB p65–induced expression of growth arrest and DNA-damage-inducible 45 beta in the Gak-KO mice. Our results suggest that loss of podocyte-associated Gak induces glomerular injury secondary to calcium dysregulation and aberrant calpain activation, which when inhibited, can provide a protective role.
Xuefei Tian, Kazunori Inoue, Yan Zhang, Ying Wang, C. John Sperati, Christopher E. Pedigo, Tingting Zhao, Meihua Yan, Marwin Groener, Dennis G. Moledina, Karen Ebenezer, Wei Li, Zhenhai Zhang, Dan A. Liebermann, Lois Greene, Peter Greer, Chirag R. Parikh, Shuta Ishibe
While autoantibodies are used in the diagnosis of rheumatoid arthritis (RA), the function of B cells in the inflamed joint remains elusive. Extensive flow cytometric characterization and SPICE algorithm analyses of single-cell synovial tissue from patients with RA revealed the accumulation of switched and double-negative memory programmed death-1 receptor–expressing (PD-1–expressing) B cells at the site of inflammation. Accumulation of memory B cells was mediated by CXCR3, evident by the observed increase in CXCR3-expressing synovial B cells compared with the periphery, differential regulation by key synovial cytokines, and restricted B cell invasion demonstrated in response to CXCR3 blockade. Notably, under 3% O2 hypoxic conditions that mimic the joint microenvironment, RA B cells maintained marked expression of MMP-9, TNF, and IL-6, with PD-1+ B cells demonstrating higher expression of CXCR3, CD80, CD86, IL-1β, and GM-CSF than their PD-1– counterparts. Finally, following functional analysis and flow cell sorting of RA PD-1+ versus PD-1– B cells, we demonstrate, using RNA-Seq and emerging fluorescence lifetime imaging microscopy of cellular NAD, a significant shift in metabolism of RA PD-1+ B cells toward glycolysis, associated with an increased transcriptional signature of key cytokines and chemokines that are strongly implicated in RA pathogenesis. Our data support the targeting of pathogenic PD-1+ B cells in RA as a focused, novel therapeutic option.
Achilleas Floudas, Nuno Neto, Viviana Marzaioli, Kieran Murray, Barry Moran, Michael G. Monaghan, Candice Low, Ronan H. Mullan, Navin Rao, Vinod Krishna, Sunil Nagpal, Douglas J. Veale, Ursula Fearon
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