RESUMEN
Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.
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Proteína Potenciadora del Homólogo Zeste 2 , Neoplasias Pulmonares , Linfocitos Infiltrantes de Tumor , Neoplasias de la Mama Triple Negativas , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/inmunología , Animales , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/inmunología , Ratones , Femenino , Línea Celular Tumoral , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Microambiente Tumoral/inmunología , Proliferación Celular , Humanos , Ratones Endogámicos BALB C , Técnicas de Inactivación de Genes , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión GénicaRESUMEN
Collective invasion, the coordinated movement of cohesive packs of cells, has become recognized as a major mode of metastasis for solid tumors. These packs are phenotypically heterogeneous and include specialized cells that lead the invasive pack and others that follow behind. To better understand how these unique cell types cooperate to facilitate collective invasion, we analyzed transcriptomic sequence variation between leader and follower populations isolated from the H1299 non-small cell lung cancer cell line using an image-guided selection technique. We now identify 14 expressed mutations that are selectively enriched in leader or follower cells, suggesting a novel link between genomic and phenotypic heterogeneity within a collectively invading tumor cell population. Functional characterization of two phenotype-specific candidate mutations showed that ARP3 enhances collective invasion by promoting the leader cell phenotype and that wild-type KDM5B suppresses chain-like cooperative behavior. These results demonstrate an important role for distinct genetic variants in establishing leader and follower phenotypes and highlight the necessity of maintaining a capacity for phenotypic plasticity during collective cancer invasion.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Invasividad Neoplásica/genética , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Heterogeneidad Genética , Genómica , Humanos , Neoplasias Pulmonares/patología , Microscopía , Invasividad Neoplásica/patología , RNA-SeqRESUMEN
BACKGROUND: Intratumoral heterogeneity is defined by subpopulations with varying genotypes and phenotypes. Specialized, highly invasive leader cells and less invasive follower cells are phenotypically distinct subpopulations that cooperate during collective cancer invasion. Because leader cells are a rare subpopulation that would be missed by bulk sequencing, a novel image-guided genomics platform was used to precisely select this subpopulation. This study identified a novel leader cell mutation signature and tested its ability to predict prognosis in non-small cell lung cancer (NSCLC) patient cohorts. METHODS: Spatiotemporal genomic and cellular analysis was used to isolate and perform RNA sequencing on leader and follower populations from the H1299 NSCLC cell line, and it revealed a leader-specific mutation cluster on chromosome 16q. Genomic data from patients with lung squamous cell carcinoma (LUSC; n = 475) and lung adenocarcinoma (LUAD; n = 501) from The Cancer Genome Atlas were stratified by 16q mutation cluster (16qMC) status (16qMC+ vs 16qMC-) and compared for overall survival (OS), progression-free survival (PFS), and gene set enrichment analysis (GSEA). RESULTS: Poorer OS, poorer PFS, or both were found across all stages and among early-stage patients with 16qMC+ tumors within the LUSC and LUAD cohorts. GSEA revealed 16qMC+ tumors to be enriched for the expression of metastasis- and survival-associated gene sets. CONCLUSIONS: This represents the first leader cell mutation signature identified in patients and has the potential to better stratify high-risk NSCLC and ultimately improve patient outcomes.
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Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Linaje de la Célula/genética , Proteínas de Neoplasias/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/patología , Cromosomas Humanos Par 16/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Persona de Mediana Edad , Familia de Multigenes/genética , Mutación/genética , Invasividad Neoplásica/genética , Supervivencia sin Progresión , Análisis de Secuencia de ARNRESUMEN
Many tumors are characterized by genetic instability, producing an assortment of genetic variants of tumor cells called subclones. These tumors and their surrounding environments form complex multi-cellular ecosystems, where subclones compete for resources and cooperate to perform multiple tasks, including cancer invasion. Our recent empirical studies revealed existence of such distinct phenotypes of cancer cells, leaders and followers, in lung cancer. These two cellular subclones exchange a complex array of extracellular signals demonstrating a symbiotic relationship at the cellular level. Here, we develop a computational model of the microenvironment of the lung cancer ecosystem to explore how the interactions between subclones can advance or inhibit invasion. We found that, due to the complexity of the ecosystem, invasion may have very different dynamics characterized by the different levels of aggressiveness. By altering the signaling environment, we could alter the ecological relationship between the cell types and the overall ecosystem development. Competition between leader and follower cell populations (defined by the limited amount of resources), positive feedback within the leader cell population (controlled by the focal adhesion kinase and fibronectin signaling), and impact of the follower cells to the leaders (represented by yet undetermined proliferation signal) all had major effects on the outcome of the collective dynamics. Specifically, our analysis revealed a class of tumors (defined by the strengths of fibronectin signaling and competition) that are particularly sensitive to manipulations of the signaling environment. These tumors can undergo irreversible changes to the tumor ecosystem that outlast the manipulations of feedbacks and have a profound impact on invasive potential. Our study predicts a complex division of labor between cancer cell subclones and suggests new treatment strategies targeting signaling within the tumor ecosystem.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Modelos Biológicos , Microambiente Tumoral , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Biología Computacional , Ecosistema , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Microscopía Fluorescente , Transducción de Señal , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologíaRESUMEN
The G-protein-coupled chemokine receptor CXCR4 generates signals that lead to cell migration, cell proliferation, and other survival mechanisms that result in the metastatic spread of primary tumor cells to distal organs. Numerous studies have demonstrated that CXCR4 can form homodimers or can heterodimerize with other G-protein-coupled receptors to form receptor complexes that can amplify or decrease the signaling capacity of each individual receptor. Using biophysical and biochemical approaches, we found that CXCR4 can form an induced heterodimer with cannabinoid receptor 2 (CB2) in human breast and prostate cancer cells. Simultaneous, agonist-dependent activation of CXCR4 and CB2 resulted in reduced CXCR4-mediated expression of phosphorylated ERK1/2 and ultimately reduced cancer cell functions such as calcium mobilization and cellular chemotaxis. Given that treatment with cannabinoids has been shown to reduce invasiveness of cancer cells as well as CXCR4-mediated migration of immune cells, it is plausible that CXCR4 signaling can be silenced through a physical heterodimeric association with CB2, thereby inhibiting subsequent functions of CXCR4. Taken together, the data illustrate a mechanism by which the cannabinoid system can negatively modulate CXCR4 receptor function and perhaps tumor progression.
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Neoplasias de la Mama/patología , Neoplasias de la Próstata/patología , Multimerización de Proteína , Receptor Cannabinoide CB2/química , Receptor Cannabinoide CB2/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Animales , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Cannabinoides/farmacología , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Inmunoprecipitación , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Over the past decade image guidance systems have been widely adopted in specialties such as neurosurgery and otorhinolaryngology. Nonetheless, the evidence supporting the use of image guidance systems in surgery remains limited. New augmented reality systems offer the possibility of enhanced operating room workflow compared with existing triplanar image displays, but recent studies have highlighted several concerns, particularly the risk of inattentional blindness and impaired depth perception. The aim of this study was to compare simultaneously the effectiveness and safety of various image guidance systems against standard surgery. METHODS: In this preclinical randomised study design 50 novice surgeons were allocated to no image guidance, triplanar display, always-on solid overlay, always-on wire mesh overlay, or on-demand inverse realism overlay. Each participant was asked to identify a basilar tip aneurysm in a validated model head. The primary outcomes were time to task completion, and tool path length. The secondary outcomes were recognition of an unexpected finding (a surgical clip) and subjective depth perception (using a Likert scale). FINDINGS: Surgeons' time to task completion and tool path length were significantly lower in groups using any form of image guidance than in groups with no image guidance (p<0·001 and p=0·003, respectively). The tool path distance was also lower in groups using augmented reality than in those using triplanar display (p=0·010). Always-on solid overlay resulted in the greatest inattentional blindness (20% recognition of unexpected finding by all surgeons). Wire mesh and on-demand overlays mitigated but did not negate inattentional blindness, and were comparable with triplanar display (40% recognition of unexpected finding in all groups). Wire mesh and inverse realism overlays also resulted in better subjective depth perception than always-on solid overlay (p=0·031 and p=0·008, respectively). INTERPRETATION: This study suggests that new augmented reality platforms incorporating always-on wire mesh and on-demand inverse realism might improve surgical performance, at least in novice surgeons. All image display modalities, including existing triplanar display, carry a risk of inattentional blindness. FUNDING: Wellcome Trust.
RESUMEN
Caspase-3 is an effector caspase that is activated downstream of mitochondrial outer-membrane permeabilization (MOMP) during apoptosis. However, previous work has demonstrated that caspase-3-deficient mouse embryonic fibroblasts (MEFs) are resistant to mitochondrially mediated cell death and display a delay in the mitochondrial events of apoptosis, including Bax activation, MOMP and release of cytochrome c. Here, we show that caspase-3 regulates fibronectin secretion and impacts on cell morphology, adhesion and migration. Surprisingly, the catalytic activity of caspase-3 is not required for these non-apoptotic functions. Moreover, we found that caspase-3-deficient MEFs are not resistant to death by anoikis and that exogenous fibronectin protects wild-type MEFs from cell death induced by serum withdrawal. Taken together, our data indicate that procaspase-3 has a non-apoptotic function; it regulates the secretion of fibronectin and influences morphology, adhesion and migration. Furthermore, this novel procaspase-3 function might alter the apoptotic threshold of the cell.
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Caspasa 3/metabolismo , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Fibronectinas/metabolismo , Animales , Apoptosis , Catálisis , Supervivencia Celular/fisiología , Fibroblastos/citología , Fibroblastos/metabolismo , RatonesRESUMEN
Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of ß-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys(303) of ß-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of ß-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.
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Neoplasias de la Mama/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Witanólidos/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Regulación hacia Abajo/genética , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Huso Acromático/patología , Tubulina (Proteína)/genética , Witanólidos/farmacocinéticaRESUMEN
We have shown previously that withaferin A (WA), a bioactive component of the medicinal plant Withania somnifera, inhibits growth of cultured and xenografted human breast cancer cells and prevents breast cancer development and pulmonary metastasis incidence in a transgenic mouse model. The present study was undertaken to determine if the anticancer effect of WA involved inhibition of epithelial-mesenchymal transition (EMT). Experimental EMT induced by exposure of MCF-10A cells to tumor necrosis factor-α (TNF-α) and transforming growth factor-ß1 (TGF-ß) was partially reversed by treatment with WA but not by its structural analogs withanone or withanolide A. Combined TNF-α and TGF-ß treatments conferred partial protection against MCF-10A cell migration inhibition by WA. Inhibition of TNF-α and TGF-ß-induced MCF-10A cell migration by WA exposure was modestly attenuated by knockdown of E-cadherin protein. MCF-7 and MDA-MB-231 cells exposed to WA exhibited sustained (MCF-7) or transient (MDA-MB-231) induction of E-cadherin protein. On the other hand, the level of vimentin protein was increased markedly after 24 h treatment of MDA-MB-231 cells with WA. WA-induced apoptosis was not affected by vimentin protein knockdown in MDA-MB-231 cells. Protein level of vimentin was significantly lower in the MDA-MB-231 xenografts as well as in MMTV-neu tumors from WA-treated mice compared with controls. The major conclusions of the present study are that (a) WA treatment inhibits experimental EMT in MCF-10A cells, and (b) mammary cancer growth inhibition by WA administration is associated with suppression of vimentin protein expression in vivo.
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Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Transición Epitelial-Mesenquimal/efectos de los fármacos , Vimentina/metabolismo , Witanólidos/uso terapéutico , Animales , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Mama/efectos de los fármacos , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Ratones Desnudos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vimentina/análisis , Witanólidos/farmacologíaRESUMEN
Liver kinase ß1 (LKB1, also known as STK11) is a serine/threonine kinase that has multiple cellular functions including the regulation of cell polarity and motility. Murine proteomic studies show that LKB1 loss causes aberrant adhesion signaling; however, the mechanistic underpinnings of this relationship are unknown. We show that cells stably depleted of LKB1 or its co-activator STRADα have increased phosphorylation of focal adhesion kinase (FAK) at Tyr(397)/Tyr(861) and enhanced adhesion to fibronectin. LKB1 associates in a complex with FAK and LKB1 accumulation at the cellular leading edge is mutually excluded from regions of activated Tyr(397)-FAK. LKB1-compromised cells lack directional persistence compared with wild-type cells, but this is restored through subsequent pharmacological FAK inhibition or depletion, showing that cell directionality is mediated through LKB1-FAK signaling. Live cell confocal imaging reveals that LKB1-compromised cells lack normal FAK site maturation and turnover, suggesting that defects in adhesion and directional persistence are caused by aberrant adhesion dynamics. Furthermore, re-expression of full-length wild-type or the LKB1 N-terminal domain repressed FAK activity, whereas the kinase domain or C-terminal domain alone did not, indicating that FAK suppression is potentially regulated through the LKB1 N-terminal domain. Based upon these results, we conclude that LKB1 serves as a FAK repressor to stabilize focal adhesion sites, and when LKB1 function is compromised, aberrant FAK signaling ensues, resulting in rapid FAK site maturation and poor directional persistence.
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Movimiento Celular , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , Quinasas de la Proteína-Quinasa Activada por el AMP , Adhesión Celular , Línea Celular Tumoral , Polaridad Celular , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/genética , Adhesiones Focales/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/química , Quinolonas/farmacología , ARN Interferente Pequeño/genética , Análisis de la Célula Individual , Sulfonas/farmacologíaRESUMEN
Phenotypic heterogeneity poses a significant hurdle for cancer treatment but is under-characterized in the context of tumor invasion. Amidst the range of phenotypic heterogeneity across solid tumor types, collectively invading cells and single cells have been extensively characterized as independent modes of invasion, but their intercellular interactions have rarely been explored. Here, we isolated collectively invading cells and single cells from the heterogeneous 4T1 cell line and observed extensive transcriptional and epigenetic diversity across these subpopulations. By integrating these datasets, we identified laminin-332 as a protein complex exclusively secreted by collectively invading cells. Live-cell imaging revealed that laminin-332 derived from collectively invading cells increased the velocity and directionality of single cells. Despite collectively invading and single cells having similar expression of the integrin α6ß4 dimer, single cells demonstrated higher Rac1 activation upon laminin-332 binding to integrin α6ß4. This mechanism suggests a novel commensal relationship between collectively invading and single cells, wherein collectively invading cells promote the invasive potential of single cells through a laminin-332/Rac1 axis.
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Laminina , Proteína de Unión al GTP rac1 , Humanos , Movimiento Celular , Integrina alfa6beta4/genética , Kalinina , Laminina/genética , Laminina/metabolismo , Neoplasias/genética , Simbiosis , Animales , Ratones , Línea Celular Tumoral , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Targeting cancer metabolism to limit cellular energy and metabolite production is an attractive therapeutic approach. Here, we developed analogs of the bisbiguanide, alexidine, to target lung cancer cell metabolism and assess a structure-activity relationship (SAR). The SAR led to the identification of two analogs, AX-4 and AX-7, that limit cell growth via G1/G0 cell-cycle arrest and are tolerated in vivo with favorable pharmacokinetics. Mechanistic evaluation revealed that AX-4 and AX-7 induce potent mitochondrial defects; mitochondrial cristae were deformed and the mitochondrial membrane potential was depolarized. Additionally, cell metabolism was rewired, as indicated by reduced oxygen consumption and mitochondrial ATP production, with an increase in extracellular lactate. Importantly, AX-4 and AX-7 impacted overall cell behavior, as these compounds reduced collective cell invasion. Taken together, our study establishes a class of bisbiguanides as effective mitochondria and cell invasion disrupters, and proposes bisbiguanides as promising approaches to limiting cancer metastasis.
RESUMEN
LKB1 is a Ser/Thr kinase, and its activity is regulated by the pseudokinase, STE20-related adaptor α (STRADα). The STRADα-LKB1 pathway plays critical roles in epithelial cell polarity, neuronal polarity, and cancer metastasis. Though much attention is given to the STRADα-LKB1 pathway, the function of STRADα itself, including a role outside of the LKB1 pathway, has not been well-studied. Data in Caenorhabditis elegans suggest that STRADα has an LKB1-independent role in regulating cell polarity, and therefore we tested the hypothesis that STRADα regulates cancer cell polarity and motility when wild-type LKB1 is absent. These results show that STRADα protein is reduced in LKB1-null cell lines (mutation or homozygous deletion) and this partial degradation occurs through the Hsp90-dependent proteasome pathway. The remaining STRADα participates in cell polarity and invasion, such that STRADα depletion results in misaligned lamellipodia, improper Golgi positioning, and reduced invasion. To probe the molecular basis of this defect, we show that STRADα associates in a complex with PAK1, and STRADα loss disrupts PAK1 activity via Thr(423) PAK1 phosphorylation. When STRADα is depleted, PAK1-induced invasion could not occur, suggesting that STRADα is necessary for PAK1 to drive motility. Furthermore, STRADα overexpression caused increased activity of the PAK1-activating protein, rac1, and a constitutively active rac1 mutant (Q61L) rescued pPAK(Thr423) and STRADα invasion defects. Taken together, these results show that a STRADα-rac1-PAK1 pathway regulates cell polarity and invasion in LKB1-null cells. It also suggests that while the function of LKB1 and STRADα undoubtedly overlap, they may also have mutually exclusive roles.
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Polaridad Celular/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Secuencia de Bases , Línea Celular , Cartilla de ADN , HumanosRESUMEN
Mammalian target of rapamycin (mTOR) is aberrantly activated in many cancer types, and two rapamycin derivatives are currently approved by the Food and Drug Administration (FDA) of the United States for treating renal cell carcinoma. Mechanistically, mTOR is hyperactivated in human cancers either due to the genetic activation of its upstream activating signaling pathways or the genetic inactivation of its negative regulators. The tumor suppressor liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is involved in cell polarity, cell detachment and adhesion, tumor metastasis, and energetic stress response. A key role of LKB1 is to negatively regulate the activity of mTOR complex 1 (mTORC1). This review summarizes the molecular basis of this negative interaction and recent research progress in this area.
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Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Animales , Antibióticos Antineoplásicos/uso terapéutico , Modelos Animales de Enfermedad , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/metabolismo , Femenino , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirolimus/uso terapéutico , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The cornerstone of stroke care is expedient management that varies depending on the time since stroke onset. Consequently, clinical decision making is centered on accurate knowledge of timing and often requires a radiologist to interpret Computed Tomography (CT) of the brain to confirm the occurrence and age of an event. These tasks are particularly challenging due to the subtle expression of acute ischemic lesions and the dynamic nature of their appearance. Automation efforts have not yet applied deep learning to estimate lesion age and treated these two tasks independently, so, have overlooked their inherent complementary relationship. To leverage this, we propose a novel end-to-end multi-task transformer-based network optimized for concurrent segmentation and age estimation of cerebral ischemic lesions. By utilizing gated positional self-attention and CT-specific data augmentation, the proposed method can capture long-range spatial dependencies while maintaining its ability to be trained from scratch under low-data regimes commonly found in medical imaging. Furthermore, to better combine multiple predictions, we incorporate uncertainty by utilizing quantile loss to facilitate estimating a probability density function of lesion age. The effectiveness of our model is then extensively evaluated on a clinical dataset consisting of 776 CT images from two medical centers. Experimental results demonstrate that our method obtains promising performance, with an area under the curve (AUC) of 0.933 for classifying lesion ages ≤ 4.5 hours compared to 0.858 using a conventional approach, and outperforms task-specific state-of-the-art algorithms.
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Neuroimagen , Accidente Cerebrovascular , Humanos , Accidente Cerebrovascular/diagnóstico por imagen , Algoritmos , Tomografía Computarizada por Rayos X , Encéfalo/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Numerous techniques have been employed to deconstruct the heterogeneity observed in normal and diseased cellular populations, including single cell RNA sequencing, in situ hybridization, and flow cytometry. While these approaches have revolutionized our understanding of heterogeneity, in isolation they cannot correlate phenotypic information within a physiologically relevant live-cell state, with molecular profiles. This inability to integrate a historical live-cell phenotype, such as invasiveness, cell:cell interactions, and changes in spatial positioning, with multi-omic data, creates a gap in understanding cellular heterogeneity. We sought to address this gap by employing lab technologies to design a detailed protocol, termed Spatiotemporal Genomics and Cellular Analysis (SaGA), for the precise imaging-based selection, isolation, and expansion of phenotypically distinct live-cells. We begin with cells stably expressing a photoconvertible fluorescent protein and employ live cell confocal microscopy to photoconvert a user-defined single cell or set of cells displaying a phenotype of interest. The total population is then extracted from its microenvironment, and the optically highlighted cells are isolated using fluorescence activated cell sorting. SaGA-isolated cells can then be subjected to multi-omics analysis or cellular propagation for in vitro or in vivo studies. This protocol can be applied to a variety of conditions, creating protocol flexibility for user-specific research interests. The SaGA technique can be accomplished in one workday by non-specialists and results in a phenotypically defined cellular subpopulation for integration with multi-omics techniques. We envision this approach providing multi-dimensional datasets exploring the relationship between live-cell phenotype and multi-omic heterogeneity within normal and diseased cellular populations.