RESUMEN
Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis.
High-grade serous ovarian cancer (or HGSOC for short) is the fifth leading cause of cancer-related deaths in women. It is generally diagnosed at an advanced stage of disease when the cancer has already spread to other parts of the body. Surgical removal of tumors and subsequent treatment with chemotherapy often reduces the signs and symptoms of the disease for a time but some cancer cells tend to survive so that patients eventually relapse. The HGSOC cells typically spread from the ovaries by moving through the liquid surrounding organs in the abdomen. The cells clump together and enter an inactive state known as dormancy that allows them to survive chemotherapy and low-nutrient conditions. Understanding how to develop new drug therapies that target dormant cancer cells is thought to be an important step in prolonging the life of HGSOC patients. Cancer cells are hardwired to multiply and grow, so Perampalam et al. reasoned that becoming dormant poses challenges for HGSOC cells, which may create unique vulnerabilities not shared by proliferating cancer cells. To find out more, the researchers used HGSOC cells that had been isolated from patients and grown in the laboratory. The team used a gene editing technique to screen HGSOC cells for genes required by the cells to survive when they are dormant. The experiments found that genes involved in a cell signaling pathway, known as Netrin signaling, were critical for the cells to survive. Previous studies have shown that Netrin signaling helps the nervous system form in embryos and inhibits a program of controlled cell death in some cancers. Perampalam et al. discovered that Netrins were present in the environment immediately surrounding dormant HGSOC cells. Human HGSOC patients with higher levels of Netrin gene expression had poorer prognoses than patients with lower levels of Netrin gene expression. Further experiments demonstrated that Netrins help dormant HGSOC cells to spread around the body. These findings suggest that Netrin signalling may provide useful targets for future drug therapies against dormant cells in some ovarian cancers. This could include repurposing drugs already in development or creating new inhibitors of this pathway.
Asunto(s)
Carcinoma Epitelial de Ovario , Supervivencia Celular , Netrinas , Neoplasias Ováricas , Transducción de Señal , Humanos , Femenino , Animales , Línea Celular Tumoral , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Netrinas/metabolismo , Netrinas/genética , Ratones , Netrina-1/metabolismo , Netrina-1/genética , Proliferación Celular , Receptores de Netrina/metabolismo , Receptores de Netrina/genéticaRESUMEN
Mammalian cells repress expression of repetitive genomic sequences by forming heterochromatin. However, the consequences of ectopic repeat expression remain unclear. Here we demonstrate that inhibitors of EZH2, the catalytic subunit of the Polycomb repressive complex 2 (PRC2), stimulate repeat misexpression and cell death in resting splenic B cells. B cells are uniquely sensitive to these agents because they exhibit high levels of histone H3 lysine 27 trimethylation (H3K27me3) and correspondingly low DNA methylation at repeat elements. We generated a pattern recognition receptor loss-of-function mouse model, called RIC, with mutations in Rigi (encoding for RIG-I), Ifih1 (MDA5), and Cgas. In both wildtype and RIC mutant B cells, EZH2 inhibition caused loss of H3K27me3 at repetitive elements and upregulated their expression. However, NF-κB-dependent expression of inflammatory chemokines and subsequent cell death was suppressed by the RIC mutations. We further show that inhibition of EZH2 in cancer cells requires the same pattern recognition receptors to activate an interferon response. Together, the results reveal chemokine expression induced by EZH2 inhibitors in B cells as a novel inflammatory response to genomic repeat expression. Given the overlap of genes induced by EZH2 inhibitors and Epstein-Barr virus infection, this response can be described as a form of viral mimicry.
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Linfocitos B , Proteína Potenciadora del Homólogo Zeste 2 , Infecciones por Virus de Epstein-Barr , Animales , Ratones , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Metilación de ADN , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Histonas/metabolismo , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The Farnesoid X Receptor (FXR) belongs to the nuclear receptor superfamily and is an essential bile acid (BA) receptor that regulates the expression of genes involved in the metabolism of BAs. FXR protects the liver from BA overload, which is a major etiology of hepatocellular carcinoma. Herein, we investigated the changes in gene expression and chromatin accessibility in hepatocytes by performing RNA-seq in combination with the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) using a novel FXR knockout mouse model (Fxrex5Δ: Nr1h4ex5Δ/ex5Δ) generated through CRISPR/Cas9. Consistent with previous Fxr knockout models, we found that Fxrex5Δ mice develop late-onset HCC associated with increased serum and hepatic BAs. FXR deletion was associated with a dramatic loss of chromatin accessibility, primarily at promoter-associated transcription factor binding sites. Importantly, several genes involved in BA biosynthesis and circadian rhythm were downregulated following loss of FXR, also displayed reduced chromatin accessibility at their promoter regions. Altogether, these findings suggest that FXR helps to maintain a transcriptionally active state by regulating chromatin accessibility through its binding and recruitment of transcription factors and coactivators.
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Clear evidence supports a causal link between Merkel cell polyomavirus (MCPyV) and the highly aggressive human skin cancer called Merkel cell carcinoma (MCC). Integration of viral DNA into the human genome facilitates continued expression of the MCPyV small tumor (ST) and large tumor (LT) antigens in virus-positive MCCs. In MCC tumors, MCPyV LT is truncated in a manner that renders the virus unable to replicate yet preserves the LXCXE motif that facilitates its binding to and inactivation of the retinoblastoma tumor suppressor protein (pRb). We previously developed a MCPyV transgenic mouse model in which MCC tumor-derived ST and truncated LT expression were targeted to the stratified epithelium of the skin, causing epithelial hyperplasia, increased proliferation, and spontaneous tumorigenesis. We sought to determine if any of these phenotypes required the association between the truncated MCPyV LT and pRb. Mice were generated in which K14-driven MCPyV ST/LT were expressed in the context of a homozygous RbΔLXCXE knock-in allele that attenuates LT-pRb interactions through LT's LXCXE motif. We found that many of the phenotypes including tumorigenesis that develop in the K14-driven MCPyV transgenic mice were dependent upon LT's LXCXE-dependent interaction with pRb. These findings highlight the importance of the MCPyV LT-pRb interaction in an in vivo model for MCPyV-induced tumorigenesis.
Asunto(s)
Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Infecciones Tumorales por Virus , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Antígenos Virales de Tumores/genética , Antígenos Virales de Tumores/metabolismo , Transformación Celular Neoplásica , Hiperplasia/patología , Células de Merkel/metabolismo , Células de Merkel/patología , Poliomavirus de Células de Merkel/genética , Ratones , Neoplasias Cutáneas/patologíaRESUMEN
In cancer, dormancy refers to a clinical state in which microscopic residual disease becomes non-proliferative and is largely refractory to chemotherapy. Dormancy was first described in breast cancer where disease can remain undetected for decades, ultimately leading to relapse and clinical presentation of the original malignancy. A long latency period can be explained by withdrawal from cell proliferation (cellular dormancy), or a balance between proliferation and cell death that retains low levels of residual disease (tumor mass dormancy). Research into cellular dormancy has revealed features that define this state. They include arrest of cell proliferation, altered cellular metabolism, and unique cell dependencies and interactions with the microenvironment. These characteristics can be shared by dormant cells derived from disparate primary disease sites, suggesting common features exist between them.High-grade serous ovarian cancer (HGSOC) disseminates to locations throughout the abdominal cavity by means of cellular aggregates called spheroids. These growth-arrested and therapy-resistant cells are a strong contributor to disease relapse. In this review, we discuss the similarities and differences between ovarian cancer cells in spheroids and dormant properties reported for other cancer disease sites. This reveals that elements of dormancy, such as cell cycle control mechanisms and changes to metabolism, may be similar across most forms of cellular dormancy. However, HGSOC-specific aspects of spheroid biology, including the extracellular matrix organization and microenvironment, are obligatorily disease site specific. Collectively, our critical review of current literature highlights places where HGSOC cell dormancy may offer a more tractable experimental approach to understand broad principles of cellular dormancy in cancer.
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Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.
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Envejecimiento/fisiología , Ácido Ascórbico/farmacología , Diabetes Mellitus Experimental/prevención & control , Proteína de Retinoblastoma/metabolismo , Animales , Senescencia Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Factor de Transcripción E2F1/metabolismo , Desarrollo Embrionario/genética , Femenino , Fibroblastos/efectos de los fármacos , Técnicas de Sustitución del Gen , Células Secretoras de Insulina/patología , Ratones , Fosforilación , Embarazo , Proteína de Retinoblastoma/genética , Telómero/genéticaRESUMEN
DREAM (Dp, Rb-like, E2F, and MuvB) is a transcriptional repressor complex that regulates cell proliferation, and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we used an assembly-defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys but not the brain or bone marrow. Using laser-capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting apoA-IV amyloidosis (AApoAIV). AApoAIV is a recently described form, whereby WT apoA-IV has been shown to predominate in amyloid plaques. We determined by ChIP that DREAM directly regulated Apoa4 and that the histone variant H2AZ was reduced from the Apoa4 gene body in DREAM's absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of nongenetic amyloid subtypes.
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Amiloide/metabolismo , Apolipoproteínas A/metabolismo , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/metabolismo , Complejos Multiproteicos/inmunología , Proteína p107 Similar a la del Retinoblastoma/deficiencia , Amiloide/genética , Animales , Apolipoproteínas A/genética , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/genética , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/patología , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Especificidad de Órganos/genética , Proteína p107 Similar a la del Retinoblastoma/metabolismoRESUMEN
The retinoblastoma tumor suppressor protein (RB) plays an important role in biological processes such as cell cycle control, DNA damage repair, epigenetic regulation, and genome stability. The canonical model of RB regulation is that cyclin-CDKs phosphorylate and render RB inactive in late G1/S, promoting entry into S phase. Recently, monophosphorylated RB species were described to have distinct cell-cycle-independent functions, suggesting that a phosphorylation code dictates diversity of RB function. However, a biologically relevant, functional role of RB phosphorylation at non-CDK sites has remained elusive. Here, we investigated S838/T841 dual phosphorylation, its upstream stimulus, and downstream functional output. We found that mimicking T-cell receptor activation in Jurkat leukemia cells induced sequential activation of downstream kinases including p38 MAPK and RB S838/T841 phosphorylation. This signaling pathway disrupts RB and condensin II interaction with chromatin. Using cells expressing a WT or S838A/T841A mutant RB fragment, we present evidence that deficiency for this phosphorylation event prevents condensin II release from chromatin.
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Cromatina/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética/genética , Humanos , Complejos Multiproteicos/metabolismo , Mutación/genética , Fosforilación/genética , Fosforilación/fisiología , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
BACKGROUND: The use of RNA-sequencing (RNA-seq) in molecular biology research and clinical settings has increased significantly over the past decade. Despite its widespread adoption, there is a lack of simple and interactive tools to analyze and explore RNA-seq data. Many established tools require programming or Unix/Bash knowledge to analyze and visualize results. This requirement presents a significant barrier for many researchers to efficiently analyze and present RNA-seq data. RESULTS: Here we present BEAVR, a Browser-based tool for the Exploration And Visualization of RNA-seq data. BEAVR is an easy-to-use tool that facilitates interactive analysis and exploration of RNA-seq data. BEAVR is developed in R and uses DESeq2 as its engine for differential gene expression (DGE) analysis, but assumes users have no prior knowledge of R or DESeq2. BEAVR allows researchers to easily obtain a table of differentially-expressed genes with statistical testing and then visualize the results in a series of graphs, plots and heatmaps. Users are able to customize many parameters for statistical testing, dealing with variance, clustering methods and pathway analysis to generate high quality figures. CONCLUSION: BEAVR simplifies analysis for novice users but also streamlines the RNA-seq analysis process for experts by automating several steps. BEAVR and its documentation can be found on GitHub at https://github.com/developerpiru/BEAVR. BEAVR is available as a Docker container at https://hub.docker.com/r/pirunthan/beavr.
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RNA-Seq/métodos , Programas Informáticos , Análisis por Conglomerados , Gráficos por Computador , Interpretación Estadística de Datos , HumanosRESUMEN
Interphase chromosomes are organized into topologically associated domains in order to establish and maintain integrity of transcriptional programs that remain poorly understood. Here, we show that condensin II and TFIIIC are recruited to bidirectionally transcribed promoters by a mechanism that is dependent on the retinoblastoma (RB) protein. Long-range chromosome contacts are disrupted by loss of condensin II loading, which leads to altered expression at bidirectional gene pairs. This study demonstrates that mammalian condensin II functions to organize long-range chromosome contacts and regulate transcription at specific genes. In addition, RB dependence of condensin II suggests that widespread misregulation of chromosome contacts and transcriptional alterations are a consequence of RB mutation.
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Adenosina Trifosfatasas/metabolismo , Cromosomas/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción TFIII/metabolismo , Animales , Células Cultivadas , Cromosomas/genética , Epigénesis Genética , Interfase , Ratones , Regiones Promotoras Genéticas , Activación TranscripcionalRESUMEN
Proliferative control in cancer cells is frequently disrupted by mutations in the retinoblastoma protein (RB) pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in cancer cells with RB pathway defects when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR/Cas9 in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immunocompromised mice, RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late-arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the preexisting proliferative control deficit.
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Daño del ADN , Neoplasias Pulmonares/patología , Proteínas de Unión a Retinoblastoma/genética , Eliminación de Secuencia , Ubiquitina-Proteína Ligasas/genética , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Trasplante de Neoplasias , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Two recent reports describe pharmacologic approaches to specifically treat RB1-mutant cancers. The basis of this approach is a synthetic lethal relationship between RB1 mutations and inhibition of Aurora kinases A or B.See related article by Oser et al., p. 230.See related article by Gong et al., p. 248.
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Aurora Quinasa A , Neoplasias/genética , Genes Supresores de Tumor , Humanos , Mutación , Mutaciones Letales SintéticasRESUMEN
RB-E2F transcriptional control plays a key role in regulating the timing of cell cycle progression from G1 to S-phase in response to growth factor stimulation. Despite this role, it is genetically dispensable for cell cycle exit in primary fibroblasts in response to growth arrest signals. Mice engineered to be defective for RB-E2F transcriptional control at cell cycle genes were also found to live a full lifespan with no susceptibility to cancer. Based on this background we sought to probe the vulnerabilities of RB-E2F transcriptional control defects found in Rb1R461E,K542E mutant mice (Rb1G) through genetic crosses with other mouse strains. We generated Rb1G/G mice in combination with Trp53 and Cdkn1a deficiencies, as well as in combination with KrasG12D. The Rb1G mutation enhanced Trp53 cancer susceptibility, but had no effect in combination with Cdkn1a deficiency or KrasG12D. Collectively, this study indicates that compromised RB-E2F transcriptional control is not uniformly cancer enabling, but rather has potent oncogenic effects when combined with specific vulnerabilities.
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Factores de Transcripción E2F/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Proteína de Retinoblastoma/genética , Animales , Carcinogénesis/genética , Ciclo Celular/genética , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Fibroblastos , Predisposición Genética a la Enfermedad , Humanos , Antígeno Ki-67/análisis , Ratones , Ratones Transgénicos , Mutación , Neoplasias/patología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
PD-L1 plays a central role in immune recognition of cancer cells. In this issue of Molecular Cell, Jin et al. (2019) report that a phosphorylated retinoblastoma protein contacts the DNA-binding domain of p65 NF-κB, thereby blocking transcription of PD-L1.
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Antígeno B7-H1 , Factor de Transcripción ReIA/genética , Regulación de la Expresión Génica , FN-kappa B/genética , Proteína de Retinoblastoma , Transducción de SeñalRESUMEN
BACKGROUND: Brain metastasis is becoming increasingly prevalent in breast cancer due to improved extra-cranial disease control. With emerging availability of modern image-guided radiation platforms, mouse models of brain metastases and small animal magnetic resonance imaging (MRI), we examined brain metastases' responses from radiotherapy in the pre-clinical setting. In this study, we employed half brain irradiation to reduce inter-subject variability in metastases dose-response evaluations. METHODS: Half brain irradiation was performed on a micro-CT/RT system in a human breast cancer (MDA-MB-231-BR) brain metastasis mouse model. Radiation induced DNA double stranded breaks in tumors and normal mouse brain tissue were quantified using γ-H2AX immunohistochemistry at 30 min (acute) and 11 days (longitudinal) after half-brain treatment for doses of 8, 16 and 24 Gy. In addition, tumor responses were assessed volumetrically with in-vivo longitudinal MRI and histologically for tumor cell density and nuclear size. RESULTS: In the acute setting, γ-H2AX staining in tumors saturated at higher doses while normal mouse brain tissue continued to increase linearly in the phosphorylation of H2AX. While γ-H2AX fluorescence intensities returned to the background level in the brain 11 days after treatment, the residual γ-H2AX phosphorylation in the radiated tumors remained elevated compared to un-irradiated contralateral tumors. With radiation, MRI-derived relative tumor growth was significantly reduced compared to the un-irradiated side. While there was no difference in MRI tumor volume growth between 16 and 24 Gy, there was a significant reduction in tumor cell density from histology with increasing dose. In the longitudinal study, nuclear size in the residual tumor cells increased significantly as the radiation dose was increased. CONCLUSIONS: Radiation damages to the DNAs in the normal brain parenchyma are resolved over time, but remain unrepaired in the treated tumors. Furthermore, there is a radiation dose response in nuclear size of surviving tumor cells. Increase in nuclear size together with unrepaired DNA damage indicated that the surviving tumor cells post radiation had continued to progress in the cell cycle with DNA replication, but failed cytokinesis. Half brain irradiation provides efficient evaluation of dose-response for cancer cell lines, a pre-requisite to perform experiments to understand radio-resistance in brain metastases.
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Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/secundario , Encéfalo/efectos de la radiación , Neoplasias de la Mama/patología , Irradiación Craneana/métodos , Modelos Animales de Enfermedad , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Neoplasias Encefálicas/diagnóstico por imagen , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Femenino , Histonas/metabolismo , Histonas/efectos de la radiación , Humanos , Estudios Longitudinales , Ratones , Ratones Desnudos , Fosforilación/efectos de la radiación , Tolerancia a Radiación/fisiología , Dosificación Radioterapéutica , Rayos XRESUMEN
The canonical model of RB-mediated tumour suppression developed over the past 30 years is based on the regulation of E2F transcription factors to restrict cell cycle progression. Several additional functions have been proposed for RB, on the basis of which a non-canonical RB pathway can be described. Mechanistically, the non-canonical RB pathway promotes histone modification and regulates chromosome structure in a manner distinct from cell cycle regulation. These functions have implications for chemotherapy response and resistance to targeted anticancer agents. This Opinion offers a framework to guide future studies of RB in basic and clinical research.
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Estructuras Cromosómicas , Resistencia a Antineoplásicos , Código de Histonas , Neoplasias/metabolismo , Proteína de Retinoblastoma/metabolismo , Antineoplásicos/uso terapéutico , Proliferación Celular , Cromatina/metabolismo , Reparación del ADN/fisiología , Factores de Transcripción E2F/metabolismo , Epigénesis Genética , Inestabilidad Genómica , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , FosforilaciónRESUMEN
The retinoblastoma protein (pRB) plays a key role in proliferative control and genome stability. For these reasons its functions are considered to be tumor suppressive. Its functional status offers critical insight into proliferative control signaling in tissues and in developing malignancies. In this chapter, we outline basic procedures to detect the retinoblastoma protein in formalin fixed, paraffin embedded tissue sections. In addition, we provide protocols to detect phosphorylation levels of pRB in tissues and offer controls to ensure fidelity of measurement. Importantly, these staining methods utilize broadly available reagents and equipment making them accessible to most biomedical research laboratories.
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Técnicas para Inmunoenzimas/métodos , Neoplasias/diagnóstico , Proteínas de Unión a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Formaldehído , Humanos , Neoplasias/metabolismo , Adhesión en Parafina , FosforilaciónRESUMEN
Organization of chromatin structure is indispensible to the maintenance of genome integrity. The retinoblastoma tumor suppressor protein (pRB) mediates both transcriptional repression and chromatin organization, but the independent contributions of these functions have been difficult to study. Here, we utilize a synthetic Rb1 mutant allele (F832A) that maintains pRB association at cell cycle gene promoters, but disrupts a cyclin-dependent kinase (CDK)-resistant interaction with E2F1 to reduce occupancy of pRB on intergenic chromatin. Reduced pRB chromatin association increases spontaneous γH2AX deposition and aneuploidy. Our data indicates that the CDK-resistant pRB-E2F1 scaffold recruits Condensin II to major satellite repeats to stabilize chromatin structure in interphase and mitosis through mechanisms that are distinct from silencing of repetitive sequence expression.
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Cromatina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Daño del ADN/genética , Mitosis/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Quinasas Ciclina-Dependientes/genética , Daño del ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Mitosis/fisiología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosforilación , Proteínas de Unión a Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: The G1-S phase transition is critical to maintaining proliferative control and preventing carcinogenesis. The retinoblastoma tumor suppressor is a key regulator of this step in the cell cycle. RESULTS: Here we use a structure-function approach to evaluate the contributions of multiple protein interaction surfaces on pRB towards cell cycle regulation. SAOS2 cell cycle arrest assays showed that disruption of three separate binding surfaces were necessary to inhibit pRB-mediated cell cycle control. Surprisingly, mutation of some interaction surfaces had no effect on their own. Rather, they only contributed to cell cycle arrest in the absence of other pRB dependent arrest functions. Specifically, our data shows that pRB-E2F interactions are competitive with pRB-CDH1 interactions, implying that interchangeable growth arrest functions underlie pRB's ability to block proliferation. Additionally, disruption of similar cell cycle control mechanisms in genetically modified mutant mice results in ectopic DNA synthesis in the liver. CONCLUSIONS: Our work demonstrates that pRB utilizes a network of mechanisms to prevent cell cycle entry. This has important implications for the use of new CDK4/6 inhibitors that aim to activate this proliferative control network.
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Epithelial ovarian cancer (EOC) generates multicellular aggregates called spheroids that detach from the primary tumor and disseminate through ascites. Spheroids possess a number of characteristics of tumor dormancy including withdrawal from the cell cycle and resistance to chemotherapeutics. This report systematically analyzes the effects of RNAi depletion of 21 genes that are known to contribute to negative regulation of the cell cycle in 10 ovarian cancer cell lines. Interestingly, spheroid cell viability was compromised by loss of some cyclin-dependent kinase inhibitors such as p57Kip2, as well as Dyrk1A, Lin52, and E2F5 in most cell lines tested. Many genes essential for EOC spheroid viability are pertinent to the mammalian DREAM repressor complex. Mechanistically, the data demonstrate that DREAM is assembled upon the induction of spheroid formation, which is dependent upon Dyrk1A. Loss of Dyrk1A results in retention of the b-Myb-MuvB complex, elevated expression of DREAM target genes, and increased DNA synthesis that is coincident with cell death. Inhibition of Dyrk1A activity using pharmacologic agents Harmine and INDY compromises viability of spheroids and blocks DREAM assembly. In addition, INDY treatment improves the response to carboplatin, suggesting this is a therapeutic target for EOC treatment.Implications: Loss of negative growth control mechanisms in cancer dormancy lead to cell death and not proliferation, suggesting they are an attractive therapeutic approach. Mol Cancer Res; 15(4); 371-81. ©2016 AACR.