RESUMO
Studies in cultured neurons have shown that neurofilaments are cargoes of axonal transport that move rapidly but intermittently along microtubule tracks. However, the extent to which axonal neurofilaments move in vivo has been controversial. Some researchers have proposed that most axonally transported neurofilaments are deposited into a persistently stationary network and that only a small proportion of axonal neurofilaments are transported in mature axons. Here we use the fluorescence photoactivation pulse-escape technique to test this hypothesis in intact peripheral nerves of adult male hThy1-paGFP-NFM mice, which express low levels of mouse neurofilament protein M tagged with photoactivatable GFP. Neurofilaments were photoactivated in short segments of large, myelinated axons, and the mobility of these fluorescently tagged polymers was determined by analyzing the kinetics of their departure. Our results show that >80% of the fluorescence departed the window within 3 h after activation, indicating a highly mobile neurofilament population. The movement was blocked by glycolytic inhibitors, confirming that it was an active transport process. Thus, we find no evidence for a substantial stationary neurofilament population. By extrapolation of the decay kinetics, we predict that 99% of the neurofilaments would have exited the activation window after 10 h. These data support a dynamic view of the neuronal cytoskeleton in which neurofilaments cycle repeatedly between moving and pausing states throughout their journey along the axon, even in mature myelinated axons. The filaments spend a large proportion of their time pausing, but on a timescale of hours, most of them move.
Assuntos
Axônios , Filamentos Intermediários , Camundongos , Masculino , Animais , Filamentos Intermediários/metabolismo , Axônios/metabolismo , Neurônios/fisiologia , Transporte Axonal/fisiologia , Citoesqueleto/metabolismoRESUMO
The cross-sectional area of myelinated axons increases greatly during postnatal development in mammals and is an important influence on axonal conduction velocity. This radial growth is driven primarily by an accumulation of neurofilaments, which are cytoskeletal polymers that serve a space-filling function in axons. Neurofilaments are assembled in the neuronal cell body and transported into axons along microtubule tracks. The maturation of myelinated axons is accompanied by an increase in neurofilament gene expression and a decrease in neurofilament transport velocity, but the relative contributions of these processes to the radial growth are not known. Here, we address this question by computational modeling of the radial growth of myelinated motor axons during postnatal development in rats. We show that a single model can explain the radial growth of these axons in a manner consistent with published data on axon caliber, neurofilament and microtubule densities, and neurofilament transport kinetics in vivo. We find that the increase in the cross-sectional area of these axons is driven primarily by an increase in the influx of neurofilaments at early times and by a slowing of neurofilament transport at later times. We show that the slowing can be explained by a decline in the microtubule density.
Assuntos
Transporte Axonal , Filamentos Intermediários , Ratos , Animais , Filamentos Intermediários/metabolismo , Transporte Axonal/fisiologia , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Axônios/metabolismo , Citoesqueleto/metabolismo , Mamíferos/metabolismoRESUMO
Growth and metastasis of colorectal cancer is closely connected to the biosynthetic capacity of tumor cells, and colorectal cancer stem cells that reside at the top of the intratumoral hierarchy are especially dependent on this feature. By performing disease modeling on patient-derived tumor organoids, we found that elevated expression of the ribosome biogenesis factor NLE1 occurs upon SMAD4 loss in TGFß1-exposed colorectal cancer organoids. TGFß signaling-mediated downregulation of NLE1 was prevented by ectopic expression of c-MYC, which occupied an E-box-containing region within the NLE1 promoter. Elevated levels of NLE1 were found in colorectal cancer cohorts compared with normal tissues and in colorectal cancer subtypes characterized by Wnt/MYC and intestinal stem cell gene expression. In colorectal cancer cells and organoids, NLE1 was limiting for de novo protein biosynthesis. Upon NLE1 ablation, colorectal cancer cell lines activated p38/MAPK signaling, accumulated p62- and LC3-positive structures indicative of impaired autophagy, and displayed more reactive oxygen species. Phenotypically, knockout of NLE1 inhibit.ed proliferation, migration and invasion, clonogenicity, and anchorage-independent growth. NLE1 loss also increased the fraction of apoptotic tumor cells, and deletion of TP53 further sensitized NLE1-deficient colorectal cancer cells to apoptosis. In an endoscopy-guided orthotopic mouse transplantation model, ablation of NLE1 impaired tumor growth in the colon and reduced primary tumor-derived liver metastasis. In patients with colorectal cancer, NLE1 mRNA levels predicted overall and relapse-free survival. Taken together, these data reveal a critical role of NLE1 in colorectal cancer growth and progression and suggest that NLE1 represents a potential therapeutic target in colorectal cancer patients. SIGNIFICANCE: NLE1 limits de novo protein biosynthesis and the tumorigenic potential of advanced colorectal cancer cells, suggesting NLE1 could be targeted to improve the treatment of metastatic colorectal cancer.
Assuntos
Neoplasias Colorretais , Genes myc , Proteínas dos Microfilamentos , Proteína Smad4 , Animais , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Proteínas dos Microfilamentos/genética , Biossíntese de Proteínas , Proteína Smad4/genética , Regulação para Cima , HumanosRESUMO
Block-sparse regularization is already well known in active thermal imaging and is used for multiple-measurement-based inverse problems. The main bottleneck of this method is the choice of regularization parameters which differs for each experiment. We show the benefits of using a learned block iterative shrinkage thresholding algorithm (LBISTA) that is able to learn the choice of regularization parameters, without the need to manually select them. In addition, LBISTA enables the determination of a suitable weight matrix to solve the underlying inverse problem. Therefore, in this paper we present LBISTA and compare it with state-of-the-art block iterative shrinkage thresholding using synthetically generated and experimental test data from active thermography for defect reconstruction. Our results show that the use of the learned block-sparse optimization approach provides smaller normalized mean square errors for a small fixed number of iterations. Thus, this allows us to improve the convergence speed and only needs a few iterations to generate accurate defect reconstruction in photothermal super-resolution imaging.
Assuntos
Algoritmos , Processamento de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador/métodos , Tomografia Computadorizada por Raios X/métodosRESUMO
Neurofilaments are abundant space-filling cytoskeletal polymers that are transported into and along axons. During postnatal development, these polymers accumulate in myelinated axons causing an expansion of axon caliber, which is necessary for rapid electrical transmission. Studies on cultured nerve cells have shown that axonal neurofilaments move rapidly and intermittently along microtubule tracks in both anterograde and retrograde directions. However, it is unclear whether neurofilament transport is also bidirectional in vivo Here, we describe a pulse-spread fluorescence photoactivation method to address this in peripheral nerves dissected from hThy1-paGFP-NFM transgenic mice, which express a photoactivatable fluorescent neurofilament protein. Neurofilaments were photoactivated in short segments of myelinated axons in tibial nerves at 2, 4, 8, and 16 weeks of age. The proximal and distal spread of the fluorescence due to the movement of the fluorescent neurofilaments was measured over time. We show that the directional bias and velocity of neurofilament transport can be calculated from these measurements. The directional bias was â¼60% anterograde and 40% retrograde and did not change significantly with age or distance along the nerve. The net velocity decreased with age and distance along the nerve, which is consistent with previous studies using radioisotopic pulse labeling. This decrease in velocity was caused by a decrease in both anterograde and retrograde movement. Thus, neurofilament transport is bidirectional in vivo, with a significant fraction of the filaments moving retrogradely in both juvenile and adult mice.
Assuntos
Transporte Axonal , Filamentos Intermediários , Animais , Transporte Axonal/fisiologia , Axônios/metabolismo , Filamentos Intermediários/metabolismo , Camundongos , Proteínas de Neurofilamentos/metabolismo , Neurônios/metabolismoRESUMO
Colorectal cancer (CRC) patient-derived organoids predict responses to chemotherapy. Here we used them to investigate relapse after treatment. Patient-derived organoids expand from highly proliferative LGR5+ tumor cells; however, we discovered that lack of optimal growth conditions specifies a latent LGR5+ cell state. This cell population expressed the gene MEX3A, is chemoresistant and regenerated the organoid culture after treatment. In CRC mouse models, Mex3a+ cells contributed marginally to metastatic outgrowth; however, after chemotherapy, Mex3a+ cells produced large cell clones that regenerated the disease. Lineage-tracing analysis showed that persister Mex3a+ cells downregulate the WNT/stem cell gene program immediately after chemotherapy and adopt a transient state reminiscent to that of YAP+ fetal intestinal progenitors. In contrast, Mex3a-deficient cells differentiated toward a goblet cell-like phenotype and were unable to resist chemotherapy. Our findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from chemotherapy and identify a candidate cell of origin of relapse after treatment in CRC.
Assuntos
Neoplasias Colorretais , Organoides , Animais , Diferenciação Celular , Neoplasias Colorretais/tratamento farmacológico , Camundongos , Células-Tronco Neoplásicas , RecidivaRESUMO
BACKGROUND: There is evidence from various models of hypoxic-ischemic injury (HII) that nitric oxide (NO) is protective. We hypothesized that either inhaled NO (iNO) or nitrite would alleviate brain injury in neonatal HII via modulation of mitochondrial function. METHODS: We tested the effects of iNO and nitrite on the Rice-Vannucci model of HII in 7-day-old rats. Brain mitochondria were isolated for flow cytometry, aconitase activity, electron paramagnetic resonance, and Seahorse assays. RESULTS: Pretreatment of pups with iNO decreased survival in the Rice-Vannucci model of HII, while iNO administered post-insult did not. MRI analysis demonstrated that pre-HII iNO at 40 ppm and post-HII iNO at 20 ppm decreased the brain lesion sizes from 6.3±1.3% to 1.0±0.4% and 1.8±0.8%, respectively. Intraperitoneal nitrite at 0.165 µg/g improved neurobehavioral performance but was harmful at higher doses and had no effect on brain infarct size. NO reacted with complex IV at the heme a3 site, decreased the oxidative stress of mitochondria challenged with anoxia and reoxygenation, and suppressed mitochondrial oxygen respiration. CONCLUSIONS: This study suggests that iNO administered following neonatal HII may be neuroprotective, possibly via its modulation of mitochondrial function.
Assuntos
Óxido Nítrico , Nitritos , Administração por Inalação , Animais , Animais Recém-Nascidos , Hipóxia , RatosRESUMO
BACKGROUND: Cancer prevention has augmented the proportion of early stage colorectal cancer (CRC) diagnoses. Especially UICC stage II CRC is in urgent need for a better patient stratification to improve clinical and therapeutic decision making. We analyzed the prognostic value of AURKA expression in stage II CRC patients. By using a scoring system based on staining intensity and frequency, we addressed the association of AURKA with patient survival and clinically relevant molecular markers. METHODS: A study cohort of 208 CRC patients (UICC stage II) was assembled. A combined measure of expression frequency and staining intensity of AURKA (H-Score) was analyzed via immunohistochemistry, and the clinical performance of this variable was studied. Association of AURKA with mutant KRAS and abundance of nuclear ß-catenin as a surrogate marker of Wnt activity was examined. Time-dependent ROC analysis revealed the prognostic performance of AURKA at different patient follow-up times. RESULTS: The AURKA H-Score correlated with good overall survival (log-rank test, p-value <0.05) and wild-type KRAS (p-value <0.01). Time-dependent ROC analysis revealed a discriminative ability of the AURKA H-Score regarding overall survival between 5 and 12 years of patient follow-up (AUC: 0.570-0.595). There was no correlation of the AURKA H-Score with disease recurrence or nuclear ß-catenin abundance. CONCLUSION: By applying universally applicable immunohistochemistry, we propose that the AURKA H-Score, which is a combined measure of staining intensity and frequency of positively staining tumor cells, correlates with good overall survival and a wild-type KRAS status in UICC stage II CRC.
Assuntos
Aurora Quinase A , Neoplasias Colorretais , Aurora Quinase A/metabolismo , Biomarcadores Tumorais/análise , Neoplasias Colorretais/patologia , Humanos , Recidiva Local de Neoplasia , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , beta Catenina/metabolismoRESUMO
We address the detection of material defects, which are inside a layered material structure using compressive sensing-based multiple-input and multiple-output (MIMO) wireless radar. Here, strong clutter due to the reflection of the layered structure's surface often makes the detection of the defects challenging. Thus, sophisticated signal separation methods are required for improved defect detection. In many scenarios, the number of defects that we are interested in is limited, and the signaling response of the layered structure can be modeled as a low-rank structure. Therefore, we propose joint rank and sparsity minimization for defect detection. In particular, we propose a non-convex approach based on the iteratively reweighted nuclear and â1-norm (a double-reweighted approach) to obtain a higher accuracy compared to the conventional nuclear norm and â1-norm minimization. To this end, an iterative algorithm is designed to estimate the low-rank and sparse contributions. Further, we propose deep learning-based parameter tuning of the algorithm (i.e., algorithm unfolding) to improve the accuracy and the speed of convergence of the algorithm. Our numerical results show that the proposed approach outperforms the conventional approaches in terms of mean squared errors of the recovered low-rank and sparse components and the speed of convergence.
Assuntos
Compressão de Dados , Processamento de Imagem Assistida por Computador , Algoritmos , Núcleo Celular , Processamento de Imagem Assistida por Computador/métodos , RadarRESUMO
BACKGROUND & AIMS: Patient-derived tumor organoids recapitulate the characteristics of colorectal cancer (CRC) and provide an ideal platform for preclinical evaluation of personalized treatment options. We aimed to model the acquisition of chemotolerance during first-line combination chemotherapy in metastatic CRC organoids. METHODS: We performed next-generation sequencing to study the evolution of KRAS wild-type CRC organoids during adaptation to irinotecan-based chemotherapy combined with epidermal growth factor receptor (EGFR) inhibition. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 protein (Cas9)-editing showed the specific effect of KRASG12D acquisition in drug-tolerant organoids. Compound treatment strategies involving Aurora kinase A (AURKA) inhibition were assessed for their capability to induce apoptosis in a drug-persister background. Immunohistochemical detection of AURKA was performed on a patient-matched cohort of primary tumors and derived liver metastases. RESULTS: Adaptation to combination chemotherapy was accompanied by transcriptomic rather than gene mutational alterations in CRC organoids. Drug-tolerant cells evaded apoptosis and up-regulated MYC (c-myelocytomatosis oncogene product)/E2F1 (E2 family transcription factor 1) and/or interferon-α-related gene expression. Introduction of KRASG12D further increased the resilience of drug-persister CRC organoids against combination therapy. AURKA inhibition restored an apoptotic response in drug-tolerant KRAS-wild-type organoids. In dual epidermal growth factor receptor (EGFR)- pathway blockade-primed CRC organoids expressing KRASG12D, AURKA inhibition augmented apoptosis in cases that had acquired increased c-MYC protein levels during chemotolerance development. In patient-matched CRC cohorts, AURKA expression was increased in primary tumors and derived liver metastases. CONCLUSIONS: Our study emphasizes the potential of patient-derived CRC organoids in modeling chemotherapy tolerance ex vivo. The applied therapeutic strategy of dual EGFR pathway blockade in combination with AURKA inhibition may prove effective for second-line treatment of chemotolerant CRC liver metastases with acquired KRAS mutation and increased AURKA/c-MYC expression.
Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Aurora Quinase A/genética , Aurora Quinase A/farmacologia , Aurora Quinase A/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Organoides/metabolismoRESUMO
Self-localization based on passive RFID-based has many potential applications. One of the main challenges it faces is the suppression of the reflected signals from unwanted objects (i.e., clutter). Typically, the clutter echoes are much stronger than the backscattered signals of the passive tag landmarks used in such scenarios. Therefore, successful tag detection can be very challenging. We consider two types of tags, namely low-Q and high-Q tags. The high-Q tag features a sparse frequency response, whereas the low-Q tag presents a broad frequency response. Further, the clutter usually showcases a short-lived response. In this work, we propose an iterative algorithm based on a low-rank plus sparse recovery approach (RPCA) to mitigate clutter and retrieve the landmark response. In addition to that, we compare the proposed approach with the well-known time-gating technique. It turns out that RPCA outperforms significantly time-gating for low-Q tags, achieving clutter suppression and tag identification when clutter encroaches on the time-gating window span, whereas it also increases the backscattered power at resonance by approximately 12 dB at 80 cm for high-Q tags. Altogether, RPCA seems a promising approach to improve the identification of passive indoor self-localization tag landmarks.
RESUMO
We present two rare cases of mixed large cell neuroendocrine carcinoma and squamous cell carcinoma of the colon. A literature search revealed only three published cases with similar histology but none of these reports provided profound molecular and mutational analyses. Our two cases exhibited a distinct, colon-like immunophenotype with strong nuclear CDX2 and ß-catenin expression in more than 90% of the tumour cells of both components. We analysed the two carcinomas regarding microsatellite stability, RAS, BRAF and PD-L1 status. In addition, next-generation panel sequencing with Ion AmpliSeq™ Cancer Hotspot Panel v2 was performed. This approach revealed mutations in FBXW7, CTNNB1 and PIK3CA in the first case and FBXW7 and RB1 mutations in the second case. We looked for similar mutational patterns in three publicly available colorectal adenocarcinoma data sets, as well as in collections of colorectal mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs) and colorectal neuroendocrine carcinomas. This approach indicated that the FBXW7 point mutation, without being accompanied by classical adenoma-carcinoma sequence mutations, such as APC, KRAS and TP53, likely occurs at a relatively high frequency in mixed neuroendocrine and squamous cell carcinoma and therefore may be characteristic for this rare tumour type. FBXW7 codifies the substrate recognition element of an ubiquitin ligase, and inactivating FBXW7 mutations lead to an exceptional accumulation of its target ß-catenin which results in overactivation of the Wnt-signalling pathway. In line with previously described hypotheses of de-differentiation of colon cells by enhanced Wnt-signalling, our data indicate a crucial role for mutant FBXW7 in the unusual morphological switch that determines these rare neoplasms. Therefore, mixed large cell neuroendocrine and a squamous cell carcinoma can be considered as a distinct carcinoma entity in the colon, defined by morphology, immunophenotype and distinct molecular genetic alteration(s).
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Grandes/genética , Carcinoma Neuroendócrino/genética , Carcinoma de Células Escamosas/genética , Neoplasias do Colo/genética , Proteína 7 com Repetições F-Box-WD/genética , Mutação , Neoplasias Complexas Mistas/genética , Biomarcadores Tumorais/análise , Carcinoma de Células Grandes/química , Carcinoma de Células Grandes/patologia , Carcinoma de Células Grandes/terapia , Carcinoma Neuroendócrino/química , Carcinoma Neuroendócrino/patologia , Carcinoma Neuroendócrino/terapia , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Neoplasias do Colo/química , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Evolução Fatal , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Complexas Mistas/química , Neoplasias Complexas Mistas/patologia , Neoplasias Complexas Mistas/terapia , Fenótipo , Resultado do TratamentoRESUMO
A photothermal super resolution technique is proposed for an improved inspection of internal defects. To evaluate the potential of the laser-based thermographic technique, an additively manufactured stainless steel specimen with closely spaced internal cavities is used. Four different experimental configurations in transmission, reflection, stepwise and continuous scanning are investigated. The applied image post-processing method is based on compressed sensing and makes use of the block sparsity from multiple measurement events. This concerted approach of experimental measurement strategy and numerical optimization enables the resolution of internal defects and outperforms conventional thermographic inspection techniques.
RESUMO
Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.
Assuntos
Transporte Axonal/fisiologia , Filamentos Intermediários/metabolismo , Modelos Teóricos , Imagem Molecular/métodos , Proteínas de Neurofilamentos/metabolismo , Neurônios/fisiologia , Nervo Tibial/metabolismo , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodosRESUMO
BACKGROUND: Wnt signaling drives epithelial self-renewal and disease progression in human colonic epithelium and colorectal cancer (CRC). Characterization of Wnt effector pathways is key for our understanding of these processes and for developing therapeutic strategies that aim to preserve tissue homeostasis. O-glycosylated cell surface proteins, such as α-dystroglycan (α-DG), mediate cellular adhesion to extracellular matrix components. We revealed a Wnt/LARGE2/α-DG signaling pathway which triggers this mode of colonic epithelial cell-to-matrix interaction in health and disease. METHODS: Next generation sequencing upon shRNA-mediated silencing of adenomatous polyposis coli (APC), and quantitative chromatin immunoprecipitation (qChIP) combined with CRISPR/Cas9-mediated transcription factor binding site targeting characterized LARGE2 as a Wnt target gene. Quantitative mass spectrometry analysis on size-fractionated, glycoprotein-enriched samples revealed functional O-glycosylation of α-DG by LARGE2 in CRC. The biology of Wnt/LARGE2/α-DG signaling was assessed by affinity-based glycoprotein enrichment, laminin overlay, CRC-to-endothelial cell adhesion, and transwell migration assays. Experiments on primary tissue, human colonic (tumor) organoids, and bioinformatic analysis of CRC cohort data confirmed the biological relevance of our findings. RESULTS: Next generation sequencing identified the LARGE2 O-glycosyltransferase encoding gene as differentially expressed upon Wnt activation in CRC. Silencing of APC, conditional expression of oncogenic ß-catenin and endogenous ß-catenin-sequestration affected LARGE2 expression. The first intron of LARGE2 contained a CTTTGATC motif essential for Wnt-driven LARGE2 expression, showed occupation by the Wnt transcription factor TCF7L2, and Wnt activation triggered LARGE2-dependent α-DG O-glycosylation and laminin-adhesion in CRC cells. Colonic crypts and organoids expressed LARGE2 mainly in stem cell-enriched subpopulations. In human adenoma organoids, activity of the LARGE2/α-DG axis was Wnt-dose dependent. LARGE2 expression was elevated in CRC and correlated with the Wnt-driven molecular subtype and intestinal stem cell features. O-glycosylated α-DG represented a Wnt/LARGE2-dependent feature in CRC cell lines and patient-derived tumor organoids. Modulation of LARGE2/α-DG signaling affected CRC cell migration through laminin-coated membranes and adhesion to endothelial cells. CONCLUSIONS: We conclude that the LARGE2 O-glycosyltransferase-encoding gene represents a direct target of canonical Wnt signaling and mediates functional O-glycosylation of α-dystroglycan (α-DG) in human colonic stem/progenitor cells and Wnt-driven CRC. Our work implies that aberrant Wnt activation augments CRC cell-matrix adhesion by increasing LARGE/α-DG-mediated laminin-adhesiveness. Video abstract.
Assuntos
Colo/patologia , Neoplasias Colorretais/metabolismo , Células Epiteliais/metabolismo , Glicosiltransferases/metabolismo , Laminina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Wnt/metabolismo , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Sequência de Bases , Adesão Celular , Diferenciação Celular , Movimento Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Distroglicanas/metabolismo , Células Endoteliais/metabolismo , Epitélio/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Glicosilação , Glicosiltransferases/genética , Células HT29 , Humanos , Intestino Delgado/metabolismo , Neoplasias Hepáticas/secundário , Proteínas de Membrana/genética , Camundongos , Organoides/metabolismo , Organoides/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Via de Sinalização WntRESUMO
Neurofilaments are abundant space-filling cytoskeletal polymers in axons that are transported along microtubule tracks. Neurofilament transport is accelerated at nodes of Ranvier, where axons are locally constricted. Strikingly, these constrictions are accompanied by sharp decreases in neurofilament number, no decreases in microtubule number, and increases in the packing density of these polymers, which collectively bring nodal neurofilaments closer to their microtubule tracks. We hypothesize that this leads to an increase in the proportion of time that the filaments spend moving and that this can explain the local acceleration. To test this, we developed a stochastic model of neurofilament transport that tracks their number, kinetic state, and proximity to nearby microtubules in space and time. The model assumes that the probability of a neurofilament moving is dependent on its distance from the nearest available microtubule track. Taking into account experimentally reported numbers and densities for neurofilaments and microtubules in nodes and internodes, we show that the model is sufficient to explain the local acceleration of neurofilaments within nodes of Ranvier. This suggests that proximity to microtubule tracks may be a key regulator of neurofilament transport in axons, which has implications for the mechanism of neurofilament accumulation in development and disease.
Assuntos
Filamentos Intermediários/metabolismo , Nós Neurofibrosos/metabolismo , Aceleração , Axônios/metabolismo , Transporte Biológico , Simulação por Computador , Fluorescência , Cinética , Microtúbulos/metabolismo , Modelos Biológicos , Bainha de Mielina/metabolismoRESUMO
Intestine is a major target of vitamin D and several studies indicate an association between vitamin D deficiency and inflammatory bowel diseases (IBD), but also increased colorectal cancer (CRC) risk and mortality. However, the putative effects of 1α,25-dihydroxyvitamin D3 (calcitriol), the active vitamin D metabolite, on human colonic stem cells are unknown. Here we show by immunohistochemistry and RNAscope in situ hybridization that vitamin D receptor (VDR) is unexpectedly expressed in LGR5+ colon stem cells in human tissue and in normal and tumor organoid cultures generated from patient biopsies. Interestingly, normal and tumor organoids respond differentially to calcitriol with profound and contrasting changes in their transcriptomic profiles. In normal organoids, calcitriol upregulates stemness-related genes, such as LGR5, SMOC2, LRIG1, MSI1, PTK7, and MEX3A, and inhibits cell proliferation. In contrast, in tumor organoids calcitriol has little effect on stemness-related genes while it induces a differentiated phenotype, and variably reduces cell proliferation. Concordantly, electron microscopy showed that calcitriol does not affect the blastic undifferentiated cell phenotype in normal organoids but it induces a series of differentiated features in tumor organoids. Our results constitute the first demonstration of a regulatory role of vitamin D on human colon stem cells, indicating a homeostatic effect on colon epithelium with relevant implications in IBD and CRC.
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Calcitriol/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Colo/citologia , Neoplasias do Colo/patologia , Organoides/citologia , Receptores de Calcitriol/metabolismo , Células-Tronco/citologia , Apoptose , Proliferação de Células , Células Cultivadas , Colo/efeitos dos fármacos , Colo/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Receptores de Calcitriol/deficiência , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
We recently described a positive feedback loop connecting c-MYC, NAMPT, DBC1 and SIRT1 that contributes to unrestricted cancer cell proliferation. Here we determine the relevance of the loop for serrated route intestinal tumorigenesis using genetically well-defined BrafV600E and K-rasG12D mouse models. In both models we show that c-MYC and SIRT1 protein expression increased through progression from hyperplasia to invasive carcinomas and metastases. It correlated with high NAMPT expression and was directly associated to activation of the oncogenic drivers. Assessing functional and molecular consequences of pharmacological interference with factors of the loop, we found that inhibition of NAMPT resulted in apoptosis and reduced clonogenic growth in human BRAF-mutant colorectal cancer cell lines and patient-derived tumoroids. Blocking SIRT1 activity was only effective when combined with a PI3K inhibitor, whereas the latter antagonized the effects of NAMPT inhibition. Interfering with the positive feedback loop was associated with down-regulation of c-MYC and temporary de-repression of TP53, explaining the anti-proliferative and pro-apoptotic effects. In conclusion we show that the c-MYC-NAMPT-DBC1-SIRT1 positive feedback loop contributes to murine serrated tumor progression. Targeting the feedback loop exerted a unique, dual therapeutic effect of oncoprotein inhibition and tumor suppressor activation. It may therefore represent a promissing target for serrated colorectal cancer, and presumably for other cancer types with deregulated c-MYC.
Assuntos
Transformação Celular Neoplásica , Citocinas/metabolismo , Neoplasias Intestinais/etiologia , Neoplasias Intestinais/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Oncogenes , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sirtuína 1/metabolismo , Animais , Apoptose/genética , Biomarcadores , Biópsia , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Imuno-Histoquímica , Neoplasias Intestinais/tratamento farmacológico , Neoplasias Intestinais/patologia , Camundongos , Mutação , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais , Sirtuína 1/antagonistas & inibidoresRESUMO
Recent advances in live cell imaging of F-actin structures, combined with pulse-chase imaging and computational modeling have suggested that actin is transported along the axon via biased polymerization of metastable actin fibers (actin trails). This mechanism is distinct from motor driven polymer transport, such as for neurofilaments and can be best described as molecular hitchhiking, where G-actin molecules are intermittently incorporated into actin fibers which grow preferentially in the anterograde direction. In this paper, we discuss how various axonal and actin trail parameters like axon diameter, trail nucleation rates, basal G-actin concentration, and trail length influence the transport rate. These predictions can help guide future experiments to verify this novel protein transport mechanism. We introduce a simplified, analytically solvable model of actin transport which relates these parameters to experimentally measurable quantities. We also discuss why a simple diffusion-based transport mechanism cannot explain bulk actin transport in the axon.
Assuntos
Actinas/metabolismo , Transporte Axonal , Axônios/fisiologia , Polimerização , Modelos Biológicos , Processos EstocásticosRESUMO
Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in neurofilament number that can be as much as 10-fold in the largest axons. To investigate how neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo Fluorescent neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of neurofilament transport (i.e., the proportion of the time that the neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains.SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of neurofilaments, which move along axons in a rapid intermittent manner. Imaging of neurofilament transport in mature myelinated axons ex vivo reveals that neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.