ABSTRACT
The extracellular matrix (ECM) undergoes substantial changes during prostate cancer (PCa) progression, thereby regulating PCa growth and invasion. Herein, a meta-analysis of multiple PCa cohorts is performed which revealed that downregulation or genomic loss of ITGA1 and ITGA2 integrin genes is associated with tumor progression and worse prognosis. Genomic deletion of both ITGA1 and ITGA2 activated epithelial-to-mesenchymal transition (EMT) in benign prostate epithelial cells, thereby enhancing their invasive potential in vitro and converting them into tumorigenic cells in vivo. Mechanistically, EMT is induced by enhanced secretion and autocrine activation of TGFß1 and nuclear targeting of YAP1. An unbiased genome-wide co-expression analysis of large PCa cohort datasets identified the transcription factor TEAD1 as a key regulator of ITGA1 and ITGA2 expression in PCa cells while TEAD1 loss phenocopied the dual loss of α1- and α2-integrins in vitro and in vivo. Remarkably, clinical data analysis revealed that TEAD1 downregulation or genomic loss is associated with aggressive PCa and together with low ITGA1 and ITGA2 expression synergistically impacted PCa prognosis and progression. This study thus demonstrated that loss of α1- and α2-integrins, either via deletion/inactivation of the ITGA1/ITGA2 locus or via loss of TEAD1, contributes to PCa progression by inducing TGFß1-driven EMT.
Subject(s)
Prostate , Prostatic Neoplasms , Male , Humans , Prostate/metabolism , Prostate/pathology , Cell Line, Tumor , Prostatic Neoplasms/genetics , Signal Transduction/genetics , Integrin alpha2/genetics , Integrin alpha2/metabolism , TEA Domain Transcription FactorsABSTRACT
BACKGROUND: Aberrant somatic genomic alteration including copy number amplification is a hallmark of cancer genomes. We previously profiled genomic landscapes of prostate cancer (PCa), yet the underlying causal genes with prognostic potential has not been defined. It remains unclear how a somatic genomic event cooperates with inherited germline variants contribute to cancer predisposition and progression. METHODS: We applied integrated genomic and clinical data, experimental models and bioinformatic analysis to identify GATA2 as a highly prevalent metastasis-associated genomic amplification in PCa. Biological roles of GATA2 in PCa metastasis was determined in vitro and in vivo. Global chromatin co-occupancy and co-regulation of GATA2 and SMAD4 was investigated by coimmunoprecipitation, ChIP-seq and RNA-seq assays. Tumor cellular assays, qRT-PCR, western blot, ChIP, luciferase assays and CRISPR-Cas9 editing methods were performed to mechanistically understand the cooperation of GATA2 with SMAD4 in promoting TGFß1 and AR signaling and mediating inherited PCa risk and progression. RESULTS: In this study, by integrated genomics and experimental analysis, we identified GATA2 as a prevalent metastasis-associated genomic amplification to transcriptionally augment its own expression in PCa. Functional experiments demonstrated that GATA2 physically interacted and cooperated with SMAD4 for genome-wide chromatin co-occupancy and co-regulation of PCa genes and metastasis pathways like TGFß signaling. Mechanistically, GATA2 was cooperative with SMAD4 to enhance TGFß and AR signaling pathways, and activated the expression of TGFß1 via directly binding to a distal enhancer of TGFß1. Strinkingly, GATA2 and SMAD4 globally mediated inherited PCa risk and formed a transcriptional complex with HOXB13 at the PCa risk-associated rs339331/6q22 enhancer, leading to increased expression of the PCa susceptibility gene RFX6. CONCLUSIONS: Our study prioritizes causal genomic amplification genes with prognostic values in PCa and reveals the pivotal roles of GATA2 in transcriptionally activating the expression of its own and TGFß1, thereby co-opting to TGFß1/SMAD4 signaling and RFX6 at 6q22 to modulate PCa predisposition and progression.
Subject(s)
Prostatic Neoplasms , Male , Humans , Prostatic Neoplasms/pathology , Prostate/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolism , Chromatin , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Smad4 Protein/genetics , Smad4 Protein/metabolism , GATA2 Transcription Factor/genetics , GATA2 Transcription Factor/metabolismABSTRACT
The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII was markedly upregulated in human breast cancer (BC) and was closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of epidermal growth factor receptor tyrosine kinase (ErbB) signaling and show that it forms a complex with ErbB1 and -2 (also known as EGFR and human epidermal growth factor receptor 2 [HER2]) and α6-integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies using Col18a1 mouse models crossed with the mouse mammary tumor virus-polyoma virus middle T antigen (MMTV-PyMT) mammary carcinogenesis model showed that ColXVIII promoted BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.
Subject(s)
Breast Neoplasms , Collagen Type XVIII , Mice , Animals , Humans , Female , Collagen Type XVIII/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptor, ErbB-2/metabolism , Cell Transformation, Neoplastic , Signal TransductionABSTRACT
Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.
Subject(s)
Actins , Microfilament Proteins , Pseudopodia , Animals , Dogs , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Adherens Junctions/metabolism , Epithelial Cells/metabolism , Intercellular Junctions/metabolism , Madin Darby Canine Kidney Cells , Membrane Proteins/metabolism , Pseudopodia/metabolism , Microfilament Proteins/metabolismABSTRACT
Collagen XVIII (ColXVIII) is a component of the extracellular matrix implicated in embryogenesis and control of tissue homoeostasis. We now provide evidence that ColXVIII has a specific role in renal branching morphogenesis as observed in analyses of total and isoform-specific knockout embryos and mice. The expression of the short and the two longer isoforms differ temporally and spatially during renal development. The lack of ColXVIII or its specific isoforms lead to congenital defects in the 3D patterning of the ureteric tree where the short isoform plays a prominent role. Moreover, the ex vivo data suggests that ColXVIII is involved in the kidney epithelial tree patterning via its N-terminal domains, and especially the Thrombospondin-1-like domain common to all isoforms. This morphogenetic function likely involves integrins expressed in the ureteric epithelium. Altogether, the results point to an important role for ColXVIII in the matrix-integrin-mediated functions regulating renal development.
Subject(s)
Collagen Type XVIII , Kidney , Protein Isoforms , Animals , Mice , Collagen Type XVIII/genetics , Collagen Type XVIII/metabolism , Integrins , Kidney/embryology , Kidney/metabolism , Morphogenesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Ureter/embryology , Ureter/metabolismABSTRACT
Genome-wide association studies have identified 270 loci conferring risk for prostate cancer (PCa), yet the underlying biology and clinical impact remain to be investigated. Here we observe an enrichment of transcription factor genes including HNF1B within PCa risk-associated regions. While focused on the 17q12/HNF1B locus, we find a strong eQTL for HNF1B and multiple potential causal variants involved in the regulation of HNF1B expression in PCa. An unbiased genome-wide co-expression analysis reveals PCa-specific somatic TMPRSS2-ERG fusion as a transcriptional mediator of this locus and the HNF1B eQTL signal is ERG fusion status dependent. We investigate the role of HNF1B and find its involvement in several pathways related to cell cycle progression and PCa severity. Furthermore, HNF1B interacts with TMPRSS2-ERG to co-occupy large proportion of genomic regions with a remarkable enrichment of additional PCa risk alleles. We finally show that HNF1B co-opts ERG fusion to mediate mechanistic and biological effects of the PCa risk-associated locus 17p13.3/VPS53/FAM57A/GEMIN4. Taken together, we report an extensive germline-somatic interaction between TMPRSS2-ERG fusion and genetic variations underpinning PCa risk association and progression.
Subject(s)
Genome-Wide Association Study , Prostatic Neoplasms , Male , Humans , Prostate , Prostatic Neoplasms/genetics , Pelvis , Germ Cells , Transcriptional Regulator ERG/genetics , Hepatocyte Nuclear Factor 1-beta/genetics , Serine Endopeptidases/genetics , Oncogene Proteins, Fusion/geneticsABSTRACT
Epithelial cell adhesion is mediated by actin cytoskeleton-linked focal adhesions (FAs) and intermediate filament-associated hemidesmosomes (HDs). HDs are formed by α6ß4-integrins and mediate stable anchoring to the extracellular matrix (ECM) while FAs containing ß1-integrins regulate cell migration. Loss of HDs has been reported in various cancers such as prostate cancer where it correlates with increased invasive migration. Here we have studied cell migration properties and FA dynamics in genetically engineered prostate epithelial cell lines with intact or disrupted HDs. Disruption of HDs by depleting α6- or ß4-integrin expression promoted collective cell migration and modulated migratory activity. Dynamic analysis of fluorescent protein-tagged FA marker proteins revealed faster FA assembly and disassembly kinetics in HD-depleted cells. FRAP analysis showed that loss of HDs correlated with faster diffusion rates of focal adhesion kinase (FAK) and vinculin in and out of FAs. These data suggest that loss of α6ß4-mediated HDs promote cell migration and FA assembly dynamics by influencing the molecular diffusion rates of FAK.
ABSTRACT
Loss of α6ß4-dependent hemidesmosomal adhesions has been observed during prostate cancer progression. However, the significance and underlying mechanisms by which aberrant hemidesmosome assembly may modulate tumorigenesis remain elusive. Using an extensive CRISPR/Cas9-mediated genetic engineering approaches in different prostate cancer cell lines combined with in vivo tumorigenesis studies in mice, bone marrow-on-chip assays and bioinformatics, as well as histological analysis of prostate cancer patient cohorts, we demonstrated that simultaneous loss of PTEN and hemidesmosomal adhesions induced several tumorigenic properties including proliferation, migration, resistance to anoikis, apoptosis, and drug treatment in vitro, and increased metastatic capacity in vivo. These effects were plectin-depended and plectin was associated with actin-rich adhesions upon hemidesmosome disruption in PTEN-negative prostate cancer cells leading to activation of EGFR/PI3K/Akt- and FAK/Src-pathways. These results suggest that analysis of PTEN and hemidesmosomal proteins may have diagnostic value helping to stratify prostate cancer patients with high risk for development of aggressive disease and highlight actin-associated plectin as a potential therapeutic target specifically in PTEN/hemidesmosome dual-negative prostate cancer.
Subject(s)
Plectin , Prostatic Neoplasms , Actins , Animals , Anoikis , Carcinogenesis , Focal Adhesions/metabolism , Humans , Male , Mice , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases , Plectin/genetics , Prostatic Neoplasms/pathologyABSTRACT
Several distinct metastasis-associated glycosylation changes have been shown to promote cancer cell invasion and metastasis, the main cause of death of cancer patients. However, it is unclear whether their presence reflects cell- or tissue-specific variations for metastasis, or species needed to drive different phases of the metastatic cascade. To address this issue from a different perspective, we investigated here whether different cancer cell lines share any glycotopes that are common and important for their invasive phenotype. By using lectin microarray glycan profiling and an established myoma tissue-based 3D invasion assay, we identified a single glycotope recognized by Helix Pomatia agglutinin (HPA), whose expression level in different cancer cells correlated significantly with their invasive potential. Lectin pull-down assay and LC-MS/MS analysis in highly- (A431 and SW-48) and poorly invasive (HepG2 and RCC4) cancer cells revealed ~85 glycoproteins of which several metastasis-promoting members of the integrin family of cell adhesion receptors, the epidermal growth factor receptor (EGFR) and the matrix metalloproteinase-14 (MMP-14) were among the abundant ones. Moreover, we showed that the level of the GalNAc glycotope in MMP-14, EGFR, αV-, ß1- and ß4 integrin in highly and poorly invasive cancer cells correlated positively with their invasive potential. Collectively, our findings suggest that altered glycosylation of several metastasis-associated glycoproteins with terminal GalNAc drives the highly invasive cancer cell phenotype.
Subject(s)
Matrix Metalloproteinase 14 , Neoplasms , Chromatography, Liquid , ErbB Receptors/metabolism , Glycoproteins/metabolism , Glycosylation , Humans , Integrin beta4/metabolism , Lectins/metabolism , Matrix Metalloproteinase 14/metabolism , Phenotype , Polysaccharides , Tandem Mass SpectrometryABSTRACT
Hemidesmosomes (HDs) are adhesive structures that ensure stable anchorage of cells to the basement membrane. They are formed by α6ß4-integrin heterodimers and linked to intermediate filaments via plectin. It has been reported that one of the most common events during the pathogenesis of prostate cancer (PCa) is the loss of HD organization. While the expression levels of ß4-integrins are strongly reduced, the expression levels of α6-integrins and plectin are maintained or even elevated, and seem to promote tumorigenic properties of PCa cells, such as proliferation, invasion, metastasis, apoptosis- and drug-resistance. In this review, we discuss the potential mechanisms of how HD components might contribute to various cellular signaling pathways to promote prostate carcinogenesis. Moreover, we summarize the current knowledge on the involvement of α6ß4-integrins and plectin in PCa initiation and progression.
ABSTRACT
Integrins are heterodimeric adhesion receptors that maintain cell-extracellular matrix (ECM) interactions in diverse tissue microenvironments. They mediate cell adhesion and signaling through the assembly of large cytoplasmic multiprotein complexes that focally connect with the cytoskeleton. Integrin adhesion complexes (IAC) are specialized by the type of integrin-ECM contact and are sensitive to mechanical forces. Thus, they encrypt context-dependent information about the microenvironment in their composition. Signals mediated through IACs modulate many aspects of cell behavior, which allows cells to adapt to their surroundings. To gain insights into their function, IACs have been isolated from cultured cells and explored by proteomics. IACs are insoluble by nature and held together by transient/weak interactions, which makes it challenging to isolate intact IACs. Usually all IACs coupled to a specified ECM, which may employ different integrins, are isolated. Here we describe an alternative method based on proximity-dependent biotin identification (BioID), where specific integrin interaction partners are labeled in live cells and isolated without the need to isolate intact IACs.
Subject(s)
Biological Assay , Carbon-Nitrogen Ligases/metabolism , Escherichia coli Proteins/metabolism , Integrin alpha Chains/metabolism , Integrin beta Chains/metabolism , Protein Interaction Mapping/methods , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Animals , Biotin/chemistry , Biotin/metabolism , Biotinylation , Carbon-Nitrogen Ligases/genetics , Cell Adhesion , Cell Membrane/chemistry , Cell Membrane/metabolism , Dogs , Escherichia coli Proteins/genetics , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Integrin alpha Chains/classification , Integrin alpha Chains/genetics , Integrin beta Chains/classification , Integrin beta Chains/genetics , Madin Darby Canine Kidney Cells , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Multimerization , Recombinant Fusion Proteins/genetics , Repressor Proteins/genetics , Staining and Labeling/methods , TransfectionABSTRACT
Cellulose nanocrystals (CNCs) are promising bio-derived nanomaterials for the bottom-up fabrication of biomedical constructs. In this report, dicarboxylic acid-functionalized CNC (DCC) was functionalized with arginylglycylaspartic acid (RGD) tripeptide as a motif for improved cell adhesion and targeting. The product (DCC-RGD) self-assembled into a more elongated nanofibrillar structure through lateral and end-to-end association. When added into poly(ethylene imine) (PEI)/pDNA polyplex solution, nanocelluloses interacted electrostatically with positively charged polyplexes without affecting their integrity. The constructs were tested for their potentials as non-viral transfection reagents. Cell viability and transfection efficiency of fibroblast NIH3T3 cells were monitored as a function of CNC concentration where, in general, viability increased as the CNC concentration increased, and transfection efficiency could be optimized. Using wild-type MDCK and αV-knockout MDCK cells, the construct was able to provide targeted uptake of polyplexes. The findings have potential applications, for example, cell-selective in vitro or ex vivo transfection of autologous mesenchymal stem cells for cell therapy, or bottom-up design of future innovative biomaterials.
Subject(s)
Cellulose/chemistry , DNA/chemistry , Imines/chemistry , Nanofibers/chemistry , Nanoparticles/chemistry , Oligopeptides/chemistry , Polyethylenes/chemistry , Animals , Cell Survival/drug effects , Cells, Cultured , Dicarboxylic Acids/chemistry , Dicarboxylic Acids/pharmacology , Dogs , Dose-Response Relationship, Drug , Madin Darby Canine Kidney Cells , Mice , Molecular Structure , NIH 3T3 Cells , Oligopeptides/pharmacology , Particle Size , Plasmids , Surface PropertiesABSTRACT
The EGFR adaptor protein, CIN85, has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill defined. Here we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase, PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SRC homology 3 domains of CIN85 interacted with the proline-arginine-rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF degradation. This activity is essential in vivo, as specific loss of the CIN85-PHD2 interaction in CRISPR/Cas9-edited cells affected growth and migration properties, as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2 and uncovered an essential survival function in tumor cells by linking growth factor adaptors with hypoxia signaling. SIGNIFICANCE: This study provides unprecedented evidence for an oxygen-independent mechanism of PHD2 regulation that has important implications in cancer cell survival. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4042/F1.large.jpg.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Triple Negative Breast Neoplasms/pathology , Adaptor Proteins, Signal Transducing/genetics , Animals , Binding Sites , Cell Line, Tumor , Female , HEK293 Cells , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Mice, Nude , Protein Interaction Domains and Motifs , Triple Negative Breast Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Deep artificial neural network learning is an emerging tool in image analysis. We demonstrate its potential in the field of digital holographic microscopy by addressing the challenging problem of determining the in-focus reconstruction depth of Madin-Darby canine kidney cell clusters encoded in digital holograms. A deep convolutional neural network learns the in-focus depths from half a million hologram amplitude images. The trained network correctly determines the in-focus depth of new holograms with high probability, without performing numerical propagation. This paper reports on extensions to preliminary work published earlier as one of the first applications of deep learning in the field of digital holographic microscopy.
ABSTRACT
Cells in tissues in vivo face a very different microenvironment than typical cultured cells plated on a plastic dish. Already several decades ago, cell biologists observed that cell lines show dramatically different morphology and growth characteristics when embedded into three-dimensional (3D) substrates or standard tissue culture plates (Montesano R, Schaller G, Orci L, Cell. 66:697-711, 1991; Barcellos-Hoff MH, Aggeler J, Ram TG, Bissell MJ, Development. 105:223-235, 1989; Simian M, Bissell MJ, J Cell Biol. 216:31-40, 2017). Despite its imminent benefit for cell biological studies, suspicion and prejudice toward more complicated sample preparation requirements limited the popularity of 3D culture techniques until recently, when it was shown that soft 3D gels made of basement membrane extracts (BME) allow prolonged culture of many types of primary epithelial cells (Clevers H, Cell. 165:1586-1597, 2016; Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al., Nature. 459:262-265, 2009). These observations have brought 3D organoid culture systems into the mainstream. Here we describe two protocols for culturing epithelial cells in 3D substrates, the "blob culture" setup where cells are fully embedded into BME gel and the "overlay setup" where cells are seeded on top of BME gel and then overlaid with a thin layer of BME (Debnath J, Brugge JS, Nat Rev Cancer. 5:675-688, 2005; Bryant DM, Datta A, Rodriguez-Fraticelli AE, Peranen J, Martin-Belmonte F, Mostov KE, Nat Cell Biol. 12:1035-1045, 2010).
Subject(s)
Organoids/cytology , Basement Membrane/metabolism , Cell Culture Techniques , Cell Line , Cells, Cultured , Epithelium , HumansABSTRACT
Integrin-mediated laminin adhesions mediate epithelial cell anchorage to basement membranes and are critical regulators of epithelial cell polarity. Integrins assemble large multiprotein complexes that link to the cytoskeleton and convey signals into the cells. Comprehensive proteomic analyses of actin network-linked focal adhesions (FA) have been performed, but the molecular composition of intermediate filament-linked hemidesmosomes (HD) remains incompletely characterized. Here we have used proximity-dependent biotin identification (BioID) technology to label and characterize the interactome of epithelia-specific ß4-integrin that, as α6ß4-heterodimer, forms the core of HDs. The analysis identified â¼150 proteins that were specifically labeled by BirA-tagged integrin-ß4. In addition to known HDs proteins, the interactome revealed proteins that may indirectly link integrin-ß4 to actin-connected protein complexes, such as FAs and dystrophin/dystroglycan complexes. The specificity of the screening approach was validated by confirming the HD localization of two candidate ß4-interacting proteins, utrophin (UTRN) and ELKS/Rab6-interacting/CAST family member 1 (ERC1). Interestingly, although establishment of functional HDs depends on the formation of α6ß4-heterodimers, the assembly of ß4-interactome was not strictly dependent on α6-integrin expression. Our survey to the HD interactome sets a precedent for future studies and provides novel insight into the mechanisms of HD assembly and function of the ß4-integrin.
Subject(s)
Integrin beta4/chemistry , Integrin beta4/metabolism , Proteomics/methods , Animals , Biotinylation , Chromatography, Liquid , Dogs , Madin Darby Canine Kidney Cells , Protein Interaction Maps , Protein Multimerization , Tandem Mass SpectrometryABSTRACT
The development of tissue fibrosis is complex and at the present time, not fully understood. Fibrosis, neurodegeneration and cerebral angiomatosis (FINCA disease) have been described in patients with mutations in NHL repeat-containing protein 2 (NHLRC2). However, the molecular functions of NHLRC2 are uncharacterized. Herein, we identified putative interacting partners for NHLRC2 using proximity-labeling mass spectrometry. We also investigated the function of NHLRC2 using immortalized cells cultured from skin biopsies of FINCA patients and normal fibroblasts with NHLRC2 knock-down and NHLRC2 overexpressing gene modifications. Transmission electron microscopy analysis of immortalized cell cultures from three FINCA patients demonstrated multilamellar bodies and distinctly organized vimentin filaments. Additionally, two of three cultures derived from patient skin biopsies contained cells that exhibited features characteristic of myofibroblasts. Altogether, the data presented in this study show for the first time that NHLRC2 is involved in cellular organization through regulation of the cytoskeleton and vesicle transport. We conclude that compound heterozygous p.Asp148Tyr and p.Arg201GlyfsTer6 mutations in NHLRC2 lead to severe tissue fibrosis in humans by enhancing the differentiation of fibroblasts to myofibroblasts.
Subject(s)
Angiomatosis/pathology , Brain Diseases/pathology , Intracellular Signaling Peptides and Proteins/metabolism , Myofibroblasts/pathology , Nerve Degeneration/genetics , Actins/genetics , Angiomatosis/genetics , Brain Diseases/genetics , Cell Differentiation/genetics , Cells, Cultured , Fibrosis , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mutation/genetics , Myofibroblasts/metabolism , Skin/metabolism , Skin/pathologyABSTRACT
There has been a growing interest toward mitochondrial fatty acid synthesis (mtFAS) since the recent discovery of a neurodegenerative human disorder termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration), which is caused by mutations in the mitochondrial enoyl-CoA/ACP (acyl carrier protein) reductase (MECR) carrying out the last step of mtFAS. We show here that MECR protein is highly expressed in mouse Purkinje cells (PCs). To elucidate mtFAS function in neural tissue, here, we generated a mouse line with a PC-specific knock-out (KO) of Mecr, leading to inactivation of mtFAS confined to this cell type. Both sexes were studied. The mitochondria in KO PCs displayed abnormal morphology, loss of protein lipoylation, and reduced respiratory chain enzymatic activities by the time these mice were 6 months of age, followed by nearly complete loss of PCs by 9 months of age. These animals exhibited balancing difficulties â¼7 months of age and ataxic symptoms were evident from 8-9 months of age on. Our data show that impairment of mtFAS results in functional and ultrastructural changes in mitochondria followed by death of PCs, mimicking aspects of the clinical phenotype. This KO mouse represents a new model for impaired mitochondrial lipid metabolism and cerebellar ataxia with a distinct and well trackable cellular phenotype. This mouse model will allow the future investigation of the feasibility of metabolite supplementation approaches toward the prevention of neurodegeneration due to dysfunctional mtFAS.SIGNIFICANCE STATEMENT We have recently reported a novel neurodegenerative disorder in humans termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration) (Heimer et al., 2016). The cause of neuron degeneration in MEPAN patients is the dysfunction of the highly conserved mitochondrial fatty acid synthesis (mtFAS) pathway due to mutations in MECR, encoding mitochondrial 2-enoyl-CoA/ACP reductase. The report presented here describes the analysis of the first mouse model suffering from mtFAS-defect-induced neurodegenerative changes due to specific disruption of the Mecr gene in Purkinje cells. Our work sheds a light on the mechanisms of neurodegeneration caused by mtFAS deficiency and provides a test bed for future treatment approaches.
Subject(s)
Cerebellum/metabolism , Fatty Acids/biosynthesis , Mitochondria/metabolism , Nerve Degeneration/metabolism , Animals , Animals, Newborn , Cerebellum/pathology , Fatty Acids/genetics , Female , HEK293 Cells , Humans , Male , Mice , Mice, Knockout , Mice, Transgenic , Mitochondria/genetics , Mitochondria/pathology , Nerve Degeneration/genetics , Nerve Degeneration/pathology , Oxidoreductases Acting on CH-CH Group Donors/biosynthesis , Oxidoreductases Acting on CH-CH Group Donors/geneticsABSTRACT
Genome-wide association studies (GWAS) have identified rs11672691 at 19q13 associated with aggressive prostate cancer (PCa). Here, we independently confirmed the finding in a cohort of 2,738 PCa patients and discovered the biological mechanism underlying this association. We found an association of the aggressive PCa-associated allele G of rs11672691 with elevated transcript levels of two biologically plausible candidate genes, PCAT19 and CEACAM21, implicated in PCa cell growth and tumor progression. Mechanistically, rs11672691 resides in an enhancer element and alters the binding site of HOXA2, a novel oncogenic transcription factor with prognostic potential in PCa. Remarkably, CRISPR/Cas9-mediated single-nucleotide editing showed the direct effect of rs11672691 on PCAT19 and CEACAM21 expression and PCa cellular aggressive phenotype. Clinical data demonstrated synergistic effects of rs11672691 genotype and PCAT19/CEACAM21 gene expression on PCa prognosis. These results provide a plausible mechanism for rs11672691 associated with aggressive PCa and thus lay the ground work for translating this finding to the clinic.
Subject(s)
Prostatic Neoplasms/genetics , RNA, Long Noncoding/genetics , RNA, Untranslated/genetics , Adult , Alleles , Cell Line, Tumor , Chromosomes, Human, Pair 19/genetics , Cohort Studies , Gene Expression Regulation, Neoplastic/genetics , Gene Frequency/genetics , Genetic Predisposition to Disease/genetics , Genome-Wide Association Study , Genotype , Homeodomain Proteins , Humans , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics , PrognosisABSTRACT
Hypoxia and loss of cell polarity are common features of malignant carcinomas. Hypoxia-inducible factor 1 (HIF1) is the major regulator of cellular hypoxia response and mediates the activation of â¼300 genes. Increased HIF1 signaling is known to be associated with epithelial-mesenchymal transformation. Here, we report that hypoxia disrupts polarized epithelial morphogenesis of MDCK cells in a HIF1α-dependent manner by modulating the transforming growth factor-ß (TGFß) signaling pathway. Analysis of potential HIF1 targets in the TGFß pathway identified the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transmembrane glycoprotein related to the type I receptors of the TGFß family, whose expression was essentially lost in HIF1-depleted cells. Similar to what was observed in HIF1-deficient cells, BAMBI-depleted cells failed to efficiently activate TGFß signaling and retained epithelial polarity during hypoxia. Taken together, we show that hypoxic conditions promote TGFß signaling in a HIF1-dependent manner and BAMBI is identified in this pathway as a novel HIF1-regulated gene that contributes to hypoxia-induced loss of epithelial polarity.