ABSTRACT
Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.
ABSTRACT
The development of the cerebral cortex involves a series of dynamic events, including cell proliferation and migration, which rely on the motor protein dynein and its regulators NDE1 and NDEL1. While the loss of function in NDE1 leads to microcephaly-related malformations of cortical development (MCDs), NDEL1 variants have not been detected in MCD patients. Here, we identified two patients with pachygyria, with or without subcortical band heterotopia (SBH), carrying the same de novo somatic mosaic NDEL1 variant, p.Arg105Pro (p.R105P). Through single-cell RNA sequencing and spatial transcriptomic analysis, we observed complementary expression of Nde1/NDE1 and Ndel1/NDEL1 in neural progenitors and post-mitotic neurons, respectively. Ndel1 knockdown by in utero electroporation resulted in impaired neuronal migration, a phenotype that could not be rescued by p.R105P. Remarkably, p.R105P expression alone strongly disrupted neuronal migration, increased the length of the leading process, and impaired nucleus-centrosome coupling, suggesting a failure in nucleokinesis. Mechanistically, p.R105P disrupted NDEL1 binding to the dynein regulator LIS1. This study identifies the first lissencephaly-associated NDEL1 variant and sheds light on the distinct roles of NDE1 and NDEL1 in nucleokinesis and MCD pathogenesis.
Subject(s)
Lissencephaly , Humans , Lissencephaly/genetics , Cell Movement/genetics , Cell Proliferation , Cerebral Cortex , Dyneins/genetics , Carrier Proteins , Microtubule-Associated Proteins/geneticsABSTRACT
BACKGROUND: CDGSH iron-sulfur domain-containing protein 2 (CISD2), a pro-longevity gene, mediates healthspan in mammals. CISD2 is down-regulated during aging. Furthermore, a persistently high level of CISD2 promotes longevity and ameliorates an age-related skin phenotype in transgenic mice. Here we translate the genetic evidence into a pharmaceutical application using a potent CISD2 activator, hesperetin, which enhances CISD2 expression in HEK001 human keratinocytes from an older person. We also treated naturally aged mice in order to study the activator's anti-aging efficacy. METHODS: We studied the biological effects of hesperetin on aging skin using, firstly, a cell-based platform, namely a HEK001 human keratinocyte cell line established from an older person. Secondly, we used a mouse model, namely old mice at 21-month old. In the latter case, we investigate the anti-aging efficacy of hesperetin on ultraviolet B (UVB)-induced photoaging and naturally aged skin. Furthermore, to identify the underlying mechanisms and potential biological pathways involved in this process we carried out transcriptomic analysis. Finally, CISD2 knockdown HEK001 keratinocytes and Cisd2 knockout mice were used to study the Cisd2-dependent effects of hesperetin on skin aging. RESULTS: Four findings are pinpointed. Firstly, in human skin, CISD2 is mainly expressed in proliferating keratinocytes from the epidermal basal layer and, furthermore, CISD2 is down-regulated in the sun-exposed epidermis. Secondly, in HEK001 human keratinocytes from an older person, hesperetin enhances mitochondrial function and protects against reactive oxygen species-induced oxidative stress via increased CISD2 expression; this enhancement is CISD2-dependent. Additionally, hesperetin alleviates UVB-induced damage and suppresses matrix metalloproteinase-1 expression, the latter being a major indicator of UVB-induced damage in keratinocytes. Thirdly, transcriptomic analysis revealed that hesperetin modulates a panel of differentially expressed genes that are associated with mitochondrial function, redox homeostasis, keratinocyte function, and inflammation in order to attenuate senescence. Intriguingly, hesperetin activates two known longevity-associated regulators, namely FOXO3a and FOXM1, in order to suppress the senescence-associated secretory phenotype. Finally, in mouse skin, hesperetin enhances CISD2 expression to ameliorate UVB-induced photoaging and this occurs via a mechanism involving CISD2. Most strikingly, late-life treatment with hesperetin started at 21-month old and lasting for 5 months, is able to retard skin aging and rejuvenate naturally aged skin in mice. CONCLUSIONS: Our results reveal that a pharmacological elevation of CISD2 expression at a late-life stage using hesperetin treatment is a feasible approach to effectively mitigating both intrinsic and extrinsic skin aging and that hesperetin could act as a functional food or as a skincare product for fighting skin aging.
Subject(s)
Hesperidin , Skin Aging , Aged , Animals , Humans , Mice , Keratinocytes , Mammals , Mice, TransgenicABSTRACT
Lissencephaly is a neurodevelopmental disorder characterized by a loss of brain surface convolutions caused by genetic variants that disrupt neuronal migration. However, the genetic origins of the disorder remain unidentified in nearly one-fifth of people with lissencephaly. Using whole-exome sequencing, we identified a de novo BAIAP2 variant, p.Arg29Trp, in an individual with lissencephaly with a posterior more severe than anterior (P>A) gradient, implicating BAIAP2 as a potential lissencephaly gene. Spatial transcriptome analysis in the developing mouse cortex revealed that Baiap2 is expressed in the cortical plate and intermediate zone in an anterior low to posterior high gradient. We next used in utero electroporation to explore the effects of the Baiap2 variant in the developing mouse cortex. We found that Baiap2 knockdown caused abnormalities in neuronal migration, morphogenesis and differentiation. Expression of the p.Arg29Trp variant failed to rescue the migration defect, suggesting a loss-of-function effect. Mechanistically, the variant interfered with the ability of BAIAP2 to localize to the cell membrane. These results suggest that the functions of BAIAP2 in the cytoskeleton, cell morphogenesis and migration are important for cortical development and for the pathogenesis of lissencephaly in humans.
Subject(s)
Lissencephaly , Animals , Humans , Mice , Brain/metabolism , Cell Movement/genetics , Cytoskeleton/metabolism , Lissencephaly/genetics , Lissencephaly/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolismABSTRACT
The high heterogeneity and low percentage of neuroendocrine cells in prostate cancer limit the utility of traditional bulk RNA sequencing and even single-cell RNA sequencing to find better biomarkers for early diagnosis and stratification. Re-clustering of specific cell-type holds great promise for identification of intra-cell-type heterogeneity. However, this has not yet been used in studying neuroendocrine prostate cancer heterogeneity. Neuroendocrine cluster(s) were individually identified in each castration-resistant prostate cancer specimen and combined for trajectory analysis. Three neuroendocrine states were identified. Neuroendocrine state 2 with the highest AR score was considered the initial starting state of neuroendocrine transdifferentiation. State 1 and state 3 with distinct high neuroendocrine scores and marker genes enriched in N-Myc and REST target genes, respectively, were considered as two different types of neuroendocrine differentiated cancer cells. These two states contained distinct groups of prostate cancer biomarkers and a strong distinguishing ability of normal versus cancerous prostate across different pathological grading was found in the N-Myc-associated state. Our data highlight the central role of N-Myc and REST in mediating lineage plasticity and classifying neuroendocrine phenotypes.
ABSTRACT
Chronic epithelial defects of the cornea, which are usually associated with severe dry eye disease, diabetes mellitus, chemical injuries or neurotrophic keratitis, as well as aging, are an unmet clinical need. CDGSH Iron Sulfur Domain 2 (CISD2) is the causative gene for Wolfram syndrome 2 (WFS2; MIM 604928). CISD2 protein is significantly decreased in the corneal epithelium of patients with various corneal epithelial diseases. Here we summarize the most updated publications and discuss the central role of CISD2 in corneal repair, as well as providing new results describing how targeting Ca2+-dependent pathways can improve corneal epithelial regeneration. This review mainly focuses on the following topics. Firstly, an overview of the cornea and of corneal epithelial wound healing. The key players involved in this process, such as Ca2+, various growth factors/cytokines, extracellular matrix remodeling, focal adhesions and proteinases, are briefly discussed. Secondly, it is well known that CISD2 plays an essential role in corneal epithelial regeneration via the maintenance of intracellular Ca2+ homeostasis. CISD2 deficiency dysregulates cytosolic Ca2+, impairs cell proliferation and migration, decreases mitochondrial function and increases oxidative stress. As a consequence, these abnormalities bring about poor epithelial wound healing and this, in turn, will lead to persistent corneal regeneration and limbal progenitor cell exhaustion. Thirdly, CISD2 deficiency induces three distinct Ca2+-dependent pathways, namely the calcineurin, CaMKII and PKCα signaling pathways. Intriguingly, inhibition of each of the Ca2+-dependent pathways seems to reverse cytosolic Ca2+ dysregulation and restore cell migration during corneal wound healing. Notably, cyclosporin, an inhibitor of calcineurin, appears to have a dual effect on both inflammatory and corneal epithelial cells. Finally, corneal transcriptomic analyses have revealed that there are six major functional groupings of differential expression genes when CISD2 deficiency is present: (1) inflammation and cell death; (2) cell proliferation, migration and differentiation; (3) cell adhesion, junction and interaction; (4) Ca2+ homeostasis; (5) wound healing and extracellular matrix; and (6) oxidative stress and aging. This review highlights the importance of CISD2 in corneal epithelial regeneration and identifies the potential of repurposing venerable FDA-approved drugs that target Ca2+-dependent pathways for new uses, namely treating chronic epithelial defects of the cornea.
Subject(s)
Calcineurin , Epithelium, Corneal , Humans , Calcineurin/metabolism , Cornea/metabolism , Epithelium, Corneal/metabolism , Signal Transduction , Wound HealingABSTRACT
BACKGROUND: Circulating tumor cells (CTCs) have been investigated as a potential biomarker for predicting prognosis and monitoring therapeutic responses in colorectal cancer (CRC). However, the sensitivity of CTCs detection is low, thus limiting the clinical utility of CTCs. We aim to examine the clinicopathological parameters that improve prognosis prediction for CRC using CTCs as a biomarker. METHODS: We enumerated CTCs in 186 CRC patients and associated the number of CTCs with the clinicopathological features and overall survival (OS) using a univariate and multivariate Cox regression model and Kaplan-Meier survival analysis. RESULTS: The presence of CTCs from 186 CRC patients was significantly associated with stage, preoperational carcinoembryonic antigen (CEA), and CA19-9 levels. Using Kaplan-Meier survival and Cox regression analysis, patients with five or more CTCs exhibited significantly worse OS compared to patients with fewer than five CTCs. The combination of CTCs with tumor marker CEA has a better OS prediction than individual CTCs or CEA and serves as a more effective prediction model in patients with CRC. CONCLUSION: We identified that patients with more than five CTCs exhibited significantly worse OS. Additionally, patients with the normal level of CEA, but who also had more than five CTCs trended towards a worse OS.
Subject(s)
Colorectal Neoplasms , Neoplastic Cells, Circulating , Humans , Prognosis , Carcinoembryonic Antigen , Neoplastic Cells, Circulating/pathology , Biomarkers, TumorABSTRACT
Tumor necrosis factor (TNF)-α is a proinflammatory cytokine involved in the pathogenesis of sarcopenia, but its short half-life and inconsistent reproducibility limit the potential of TNF-α to be an ideal sarcopenia biomarker. Anti-TNF-α, a natural consequent autoantibody to TNF-α, is an indicator of relatively prolonged TNF-α exposure, has more stable concentrations than TNF-α and should be a better alternative as a biomarker of sarcopenia. Data from 484 participants from the I-Lan Longitudinal Aging Study were used for this study, and sarcopenia was defined by the Asian Working Group for Sarcopenia 2019 consensus. Plasma levels of anti-TNF-α were determined by a sandwich ELISA approach, and levels of TNF-α were determined by an immunoassay. Compared to nonsarcopenic participants, 43 sarcopenic participants had higher levels of anti-TNF-α (0.73 ± 0.19 vs. 0.79 ± 0.25 OD, p = 0.045). Plasma levels of anti-TNF-α were positively correlated with TNF-α (r = 0.24, p < 0.001), and plasma levels of anti-TNF-α were positively correlated with adiposity (r = 0.16, p < 0.001) and negatively correlated with lean body mass (r = -0.14, p = 0.003). Individuals with increasing levels of anti-TNF-α had higher odds of being sarcopenic (OR 5.4, 95 % CI: 1.1-25.8, p = 0.035), and these associations were stronger among women and younger adults. An association between TNF-α and sarcopenia was noted only in middle-aged adults (OR 6.2, 95 % CI: 1.8-21.7, p = 0.004). Plasma anti-TNF-α levels were positively correlated with TNF-α and were significantly associated with sarcopenia. Anti-TNF-α may be a more appropriate biomarker than TNF-α for sarcopenia, but further investigations are needed to confirm its roles in sarcopenia diagnosis and treatment response evaluation.
Subject(s)
Sarcopenia , Female , Humans , Middle Aged , Aging , Biomarkers , Necrosis/complications , Reproducibility of Results , Tumor Necrosis Factor Inhibitors , Tumor Necrosis Factor-alpha/immunology , AutoantibodiesABSTRACT
Whole exome sequencing (WES) has been used to detect rare causative variants in neurological diseases. However, the efficacy of WES in genetic diagnosis of clinically heterogeneous familial stroke remains inconclusive. We prospectively searched for disease-causing variants in unrelated probands with defined familial stroke by candidate gene/hotspot screening and/or WES, depending on stroke subtypes and neuroimaging features at a referral center. The clinical significance of each variant was determined according to the American College of Medical Genetics guidelines. Among 161 probands (mean age at onset 53.2 ± 13.7 years; male 63.4%), 33 participants (20.5%) had been identified with 19 pathogenic/likely pathogenic variants (PVs; WES applied 152/161 = 94.4%). Across subtypes, the highest hit rate (HR) was intracerebral hemorrhage (ICH, 7/18 = 38.9%), particularly with the etiological subtype of structural vasculopathy (4/4 = 100%, PVs in ENG, KRIT1, PKD1, RNF213); followed by ischemic small vessel disease (SVD, 15/48 = 31.3%; PVs in NOTCH3, HTRA1, HBB). In contrast, large artery atherosclerosis (LAA, 4/44 = 9.1%) and cardioembolism (0/11 = 0%) had the lowest HR. NOTCH3 was the most common causative gene (16/161 = 9.9%), presenting with multiple subtypes of SVD (n = 13), ICH (n = 2), or LAA (n = 1). Importantly, we disclosed two previously unreported PVs, KRIT1 p.E379* in a familial cerebral cavernous malformation, and F2 p.F382L in a familial cerebral venous sinus thrombosis. The contribution of monogenic etiologies was particularly high in familial ICH and SVD subtypes in our Taiwanese cohort. Utilizing subtype-guided hotspot screening and/or subsequent WES, we unraveled monogenic causes in 20.5% familial stroke probands, including 1.2% novel PVs. Genetic diagnosis may enable early diagnosis, management and lifestyle modification. Among 161 familial stroke probands, 33 (20.5%) had been identified pathogenic or likely pathogenic monogenic variants related to stroke. The positive hit rate among all subtypes was high in intracerebral hemorrhage (ICH) and ischemic small vessel disease (SVD). Notably, two previously unreported variants, KRIT1 p.E379* in a familial cerebral cavernous malformation and F2 p.F382L in familial cerebral venous sinus thrombosis, were disclosed. CVT cerebral venous thrombosis; HTN Hypertensive subtype; LAA large artery atherosclerosis; SV structural vasculopathy; U Undetermined.
Subject(s)
Atherosclerosis , Ischemic Stroke , Sinus Thrombosis, Intracranial , Stroke , Humans , Male , Adult , Middle Aged , Aged , Exome Sequencing , Stroke/complications , Stroke/genetics , Stroke/diagnosis , Cerebral Hemorrhage/complications , Cerebral Hemorrhage/diagnostic imaging , Cerebral Hemorrhage/genetics , Atherosclerosis/complications , Ischemia/complications , Sinus Thrombosis, Intracranial/complications , Adenosine Triphosphatases , Ubiquitin-Protein LigasesABSTRACT
Matching the treatment to an individual patient's tumor state can increase therapeutic efficacy and reduce tumor recurrence. Circulating tumor cells (CTCs) derived from solid tumors are promising subjects for theragnostic analysis. To analyze how CTCs represent tumor states, we established cell lines from CTCs, primary and metastatic tumors from a mouse model and provided phenotypic and multiomic analyses of these cells. CTCs and metastatic cells, but not primary tumor cells, shared stochastic mutations and similar hypomethylation levels at transcription start sites. CTCs and metastatic tumor cells shared a hybrid epithelial/mesenchymal transcriptome state with reduced adhesive and enhanced mobilization characteristics. We tested anti-cancer drugs on tumor cells from a metastatic breast cancer patient. CTC responses mirrored the impact of drugs on metastatic rather than primary tumors. Our multiomic and clinical anti-cancer drug response results reveal that CTCs resemble metastatic tumors and establish CTCs as an ex vivo tool for personalized medicine.
ABSTRACT
PERM1 (PGC-1/ERR-induced regulator in muscle 1) is a muscle-specific protein induced by PGC-1 and ERRs. Previous studies have shown that PERM1 promotes mitochondrial biogenesis and metabolism in cardiomyocytes in vitro. However, the role of endogenous PERM1 in the heart remains to be investigated with loss-of-function studies in vivo. We report the generation and characterization of systemic Perm1 knockout (KO) mice. The baseline cardiac phenotype of the homozygous Perm1 KO mice appeared normal. However, RNA-sequencing and unbiased pathway analyses showed that homozygous downregulation of PERM1 leads to downregulation of genes involved in fatty acid and carbohydrate metabolism in the heart. Transcription factor binding site analyses suggested that PPARα and PGC-1α are involved in changes in the gene expression profile. Chromatin immunoprecipitation assays showed that PERM1 interacts with the proximal regions of PPAR response elements (PPREs) in endogenous promoters of genes involved in fatty acid oxidation. Co-immunoprecipitation and reporter gene assays showed that PERM1 promoted transcription via the PPRE, partly in a PPARα and PGC-1α dependent manner. These results suggest that endogenous PERM1 is involved in the transcription of genes involved in fatty acid oxidation through physical interaction with PPARα and PGC-1α in the heart in vivo.
Subject(s)
Lipid Metabolism , Muscle Proteins , PPAR alpha , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Animals , Fatty Acids , Mice , Mice, Knockout , Muscle Proteins/metabolism , Myocytes, Cardiac , PPAR alpha/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolismABSTRACT
Regulation of fatty acid uptake, lipid production and storage, and metabolism of lipid droplets (LDs), is closely related to lipid homeostasis, adipocyte hypertrophy and obesity. We report here that stomatin, a major constituent of lipid raft, participates in adipogenesis and adipocyte maturation by modulating related signaling pathways. In adipocyte-like cells, increased stomatin promotes LD growth or enlargements by facilitating LD-LD fusion. It also promotes fatty acid uptake from extracellular environment by recruiting effector molecules, such as FAT/CD36 translocase, to lipid rafts to promote internalization of fatty acids. Stomatin transgenic mice fed with high-fat diet exhibit obesity, insulin resistance and hepatic impairments; however, such phenotypes are not seen in transgenic animals fed with regular diet. Inhibitions of stomatin by gene knockdown or OB-1 inhibit adipogenic differentiation and LD growth through downregulation of PPARγ pathway. Effects of stomatin on PPARγ involves ERK signaling; however, an alternate pathway may also exist.
Subject(s)
Adipogenesis , Lipid Droplets , Adipogenesis/genetics , Animals , CD36 Antigens/genetics , CD36 Antigens/metabolism , Diet, High-Fat , Fatty Acids/metabolism , Lipid Droplets/metabolism , Lipid Metabolism , MAP Kinase Signaling System , Mice , Obesity/genetics , Obesity/metabolism , PPAR gamma/genetics , PPAR gamma/metabolismABSTRACT
Insular flying foxes are keystone species in island ecosystems due to their critical roles in plant pollination and seed dispersal. These species are vulnerable to population decline because of their small populations and low reproductive rates. The Formosan flying fox (Pteropus dasymallus formosus) is one of the 5 subspecies of the Ryukyu flying fox. Pteropus dasymallus formosus has suffered from a severe decline and is currently recognized as a critically endangered population in Taiwan. On the contrary, the Orii's flying fox (Pteropus dasymallus inopinatus) is a relatively stable population inhabiting Okinawa Island. Here, we applied a genomic approach called double digest restriction-site associated DNA sequencing to study these 2 subspecies for a total of 7 individuals. We detected significant genetic structure between the 2 populations. Despite their contrasting contemporary population sizes, both populations harbor very low degrees of genetic diversity. We further inferred their demographic history based on the joint folded site frequency spectrum and revealed that both P. d. formosus and P. d. inopinatus had maintained small population sizes for a long period of time after their divergence. Recently, these populations experienced distinct trajectories of demographic changes. While P. d. formosus suffered from a drastic ~10-fold population decline not long ago, P. d. inopinatus underwent a ~4.5-fold population expansion. Our results suggest separate conservation management for the 2 populations-population recovery is urgently needed for P. d. formosus while long-term monitoring for adverse genetic effects should be considered for P. d. inopinatus.
Subject(s)
Chiroptera/genetics , Genetic Variation , Genetics, Population , Animals , Conservation of Natural Resources , Endangered Species , Inbreeding , Polymorphism, Single Nucleotide , Population Density , Population Dynamics , Sequence Analysis, DNA , TaiwanABSTRACT
BACKGROUND: Obesity and its associated diseases have become a major world-wide health problem. Purple-leaf Tea (Camellia sinensis L.) (PLT), that is rich of anthocyanins, has been shown to have preventive effects on obesity and metabolic disorders. The intestinal microbiota has been shown to contribute to inflammation, obesity, and several metabolic disorders. However, whether PLT consumption could prevent obesity and diet-induced metabolic diseases by modulating the gut microbiota, is not clearly understood. METHODS: In this study, six-week-old male C57BL/6 J mice were fed a normal diet (ND) or a high fat diet (HFD) without or with PLT for 10 weeks. RESULTS: PLT modulated the gut microbiota in mice and alleviated the symptoms of HFD-induced metabolic disorders, such as insulin resistance, adipocyte hypertrophy, and hepatic steatosis. PLT increased the diversity of the microbiota and the ratio of Firmicutes to Bacteroidetes. f_Barnesiellaceae, g_Barnesiella, f_Ruminococcaceae, and f_Lachnospiraceae were discriminating faecal bacterial communities of the PLT mice that differed from the HFD mice. CONCLUSIONS: These data indicate that PLT altered the microbial contents of the gut and prevented microbial dysbiosis in the host, and consequently is involved in the modulation of susceptibility to insulin resistance, hepatic diseases, and obesity that are linked to an HFD.
Subject(s)
Adiposity/drug effects , Camellia sinensis , Fatty Liver/drug therapy , Gastrointestinal Microbiome/drug effects , Obesity/drug therapy , Plant Extracts/therapeutic use , Animals , Diet, High-Fat , Dysbiosis/etiology , Dysbiosis/prevention & control , Fatty Liver/complications , Hyperlipidemias/prevention & control , Lipid Metabolism/drug effects , Male , Mice, Inbred C57BL , Obesity/complications , Obesity/microbiology , Phytotherapy , Plant Extracts/pharmacologyABSTRACT
Glycine-N-methyl transferase (GNMT) a tumor suppressor for hepatocellular carcinoma (HCC) plays a crucial role in liver homeostasis. Its expression is downregulated in almost all the tumor tissues of HCC while the mechanism of this downregulation is not yet fully understood. Recently, we identified 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside (PGG) as a GNMT promoter enhancer compound in HCC. In this study, we aimed to delineate the mechanism by which PGG enhances GNMT expression and to investigate its effect on GNMT suppression in HCC. Microarray and pathway enrichment analysis revealed that MYC was a major target of PGG. PGG suppressed MYC mRNA and protein expression in Huh7 and Hep G2 cells in a dose- and time-dependent fashion. Furthermore, MYC expression was also reduced in xenograft tumors in PGG treated mice. Moreover, shRNA-mediated knocked-down or pharmacological inhibition of MYC resulted in a significant induction of GNMT promoter activity and endogenous GNMT mRNA expression in Huh7 cells. In contrast, overexpression of MYC significantly inhibited GNMT promoter activity and endogenous GNMT protein expression. In addition, antibodies against MYC effectively precipitated the human GNMT promoter in a chromatin immunoprecipitation assay. Lastly, GNMT expression was negatively correlated with MYC expression in human HCC samples. Interestingly, PGG not only inhibited MYC gene expression but also promoted MYC protein degradation through proteasome-independent pathways. This work reveals a novel anticancer mechanism of PGG via downregulation of MYC expression and establishes a therapeutic rationale for treatment of MYC overexpressing cancers using PGG. Our data also provide a novel mechanistic understanding of GNMT regulation through MYC in the pathogenesis of HCC.
Subject(s)
Carcinoma, Hepatocellular/pathology , Glucosides/metabolism , Glycine N-Methyltransferase/metabolism , Liver Neoplasms/pathology , Proto-Oncogene Proteins c-myc/genetics , Adult , Aged , Aged, 80 and over , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation/drug effects , Female , Glucosides/pharmacology , Glycine N-Methyltransferase/genetics , Hep G2 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Nude , Middle Aged , Promoter Regions, Genetic/genetics , Proteasome Endopeptidase Complex/metabolism , Transcriptional Activation/genetics , Xenograft Model Antitumor AssaysABSTRACT
Rapid and accurate identification of pathogen is a major quarantine strategy for outbreak prevention. We used capillary electrophoresis-random amplified polymorphic DNA (CE-RAPD) to generate highly discriminatory pathogen profiles, reduced batch effects between profiles by novel normalization procedure and pattern of technical repeats, followed by target similarity evaluation using target identification score (TIS). A full target signature contains several patterns. TIS system was optimized by training set isolates that included three species, and validated using two hundred clinical Klebsiella pneumoniae isolates. Hierarchical clustering analysis showed CE-RAPD profiles arrange clusters according to the species or the source. Moreover, samples with similar profile may display similar antibiotic susceptibility. By using a signature of four patterns, the TIS system could accurately identify target among different isolates. The variation between isolates may be caused by small change in genome. TIS system provides a standardized tool for building of outbreak firewall and facilitate data exchange.
Subject(s)
Bacterial Typing Techniques/methods , DNA, Bacterial/analysis , Random Amplified Polymorphic DNA Technique/methods , Community-Acquired Infections/genetics , Community-Acquired Infections/microbiology , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Drug Resistance, Bacterial/genetics , Electrophoresis, Capillary/methods , Electrophoresis, Gel, Pulsed-Field/methods , Escherichia coli/genetics , Escherichia coli/isolation & purification , Hospitals , Humans , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Phylogeny , Polymorphism, Restriction Fragment Length , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/isolation & purification , TranscriptomeABSTRACT
Mutations in genes involved in the production, migration, or differentiation of cortical neurons often lead to malformations of cortical development (MCDs). However, many genetic mutations involved in MCD pathogenesis remain unidentified. Here we developed a genetic screening paradigm based on transposon-mediated somatic mutagenesis by in utero electroporation and the inability of mutant neuronal precursors to migrate to the cortex and identified 33 candidate MCD genes. Consistent with the screen, several genes have already been implicated in neural development and disorders. Functional disruption of the candidate genes by RNAi or CRISPR/Cas9 causes altered neuronal distributions that resemble human cortical dysplasia. To verify potential clinical relevance of these candidate genes, we analyzed somatic mutations in brain tissue from patients with focal cortical dysplasia and found that mutations are enriched in these candidate genes. These results demonstrate that this approach is able to identify potential mouse genes involved in cortical development and MCD pathogenesis.
Subject(s)
Cerebral Cortex/abnormalities , Epilepsy/genetics , Genetic Testing/methods , Malformations of Cortical Development, Group I/genetics , Neurons/pathology , Adolescent , Adult , Animals , Biomarkers/analysis , CRISPR-Cas Systems , Cerebral Cortex/cytology , Cerebral Cortex/diagnostic imaging , Cerebral Cortex/pathology , Child , Child, Preschool , DNA Transposable Elements/genetics , Disease Models, Animal , Epilepsy/diagnosis , Epilepsy/pathology , Female , Functional Neuroimaging , Gene Knockdown Techniques , Humans , Male , Malformations of Cortical Development, Group I/diagnosis , Malformations of Cortical Development, Group I/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mutagenesis/genetics , Mutation , RNA Interference , RNA, Small Interfering/metabolism , Young AdultABSTRACT
Sarcomatoid hepatocellular carcinoma (SHC) is a rare type of HCC with significantly poorer survival than ordinary HCC. Little is known about the mechanism associated with SHC and its biomarkers and therapy. Here, we established a mouse liver cancer cell line and designated as Ymac-1. A sarcomatous appearance was observed in the allograft tumor arose from Ymac-1. Liver-secreted plasma proteins were found in Ymac-1 cultured supernatant by proteomics analysis. The positive staining of CK7, CK8, Vimentin and the suppressed expression of AFP indicated that Ymac-1 is a SHC cell line. Compared to its original tumor, an elevated level of EMT markers, N-cadherin and Vimentin, was found in Ymac-1. Ymac-1 displayed a higher migration rate and side population percentage than a mouse ordinary HCC cell line-Hepa1-6. Microarray analysis was performed to identify potential biomarkers/therapeutic targets for SHC. G6pd, a vital enzyme in pentose phosphate pathway, is highly expressed in Ymac-1. Depletion of G6pd in Ymac-1 reduced CD133 expression and sphere formation. Positive correlations between G6PD and CD133 were observed in human specimen. Higher expression of both G6PD and CD133 in tumor were associated with poor survival. In summary Ymac-1 can be a useful SHC cell model for novel biomarker and therapy development.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Hepatocellular/pathology , Cell Culture Techniques/methods , Liver Neoplasms/pathology , Animals , Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cells, Cultured , Epithelial-Mesenchymal Transition , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase/metabolism , Humans , Liver Neoplasms/metabolism , Mice , Mice, Inbred NOD , Mice, SCIDABSTRACT
Breast cancer resistance protein [BCRP/ATP-binding cassette subfamily G member 2 (ABCG2)] is a member of the ATP-binding cassette transporter family. The presence of ABCG2 on the plasma membrane in many kinds of human cancer cells contributes to multidrug resistance during chemotherapy, and it has been used as the side population marker for identifying cancer stem cells in lung cancers. We report here that, in addition to the membranous form, ABCG2 proteins are also found inside the nucleus, where they bind to the E-box of CDH1 (E-cadherin) promoter and regulate transcription of this gene. Increased expression of ABCG2 causes an increase of E-cadherin and attenuates cell migration, whereas knockdown of ABCG2 downregulates E-cadherin and enhances cell motility. In mice, xenografted A549 cells that have less ABCG2 are more likely to metastasize from the subcutaneous inoculation site to the internal organs. However, for the cancer cells that have already entered the blood circulation, an increased level of ABCG2, and correspondingly increased E-cadherin, may facilitate circulating cancer cells to colonize at a distant site and form a metastatic tumor. We propose a novel role for nuclear ABCG2 that functions as a transcription regulator and participates in modulation of cancer metastasis.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Cadherins/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Neoplasm Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Active Transport, Cell Nucleus , Animals , Antigens, CD , Cadherins/metabolism , Cell Adhesion/genetics , Cell Line, Tumor , Cell Movement/genetics , Disease Models, Animal , Heterografts , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Male , Models, Biological , Neoplasm Proteins/genetics , Neoplastic Cells, Circulating , RNA Interference , Transcription, GeneticABSTRACT
Given the significant racial and ethnic diversity in genetic variation, we are intrigued to find out whether the single nucleotide polymorphisms (SNPs) identified in genome-wide association studies of colorectal cancer (CRC) susceptibility in East Asian populations are also relevant to the population of Taiwan. Moreover, loss of heterozygosity (LOH) may provide insight into how variants alter CRC risk and how regulatory elements control gene expression. To investigate the racial and ethnic diversity of CRC-susceptibility genetic variants and their relevance to the Taiwanese population, we genotyped 705 CRC cases and 1,802 healthy controls (Taiwan Biobank) for fifteen previously reported East Asian CRC-susceptibility SNPs and four novel genetic variants identified by whole-exome sequencing. We found that rs10795668 in FLJ3802842 and rs4631962 in CCND2 were significantly associated with CRC risk in the Taiwanese population. The previously unreported rs1338565 was associated with a significant increased risk of CRC. In addition, we also genotyped tumor tissue and paired adjacent normal tissues of these 705 CRC cases to search for LOH, as well as risk-associated and protective alleles. LOH analysis revealed preferential retention of three SNPs, rs12657484, rs3802842, and rs4444235, in tumor tissues. rs4444235 has been recently reported to be a cis-acting regulator of BMP4 gene; in this study, the C allele was preferentially retained in tumor tissues (pâ=â0.0023). rs4631962 and rs10795668 contribute to CRC risk in the Taiwanese and East Asian populations, and the newly identified rs1338565 was specifically associated with CRC, supporting the ethnic diversity of CRC-susceptibility SNPs. LOH analysis suggested that the three CRC risk variants, rs12657484, rs3802842, and rs4444235, exhibited somatic allele-specific imbalance and might be critical during neoplastic progression.