RESUMEN
Glioblastoma (GBM) is the most frequent and deadly primary malignant brain tumor. Hallmarks are extensive intra-tumor and inter-tumor heterogeneity and highly invasive growth, which provide great challenges for treatment. Efficient therapy is lacking and the majority of patients survive less than 1 year from diagnosis. GBM progression and recurrence is caused by treatment-resistant glioblastoma stem cells (GSCs). GSC cultures are considered important models in target identification and drug screening studies. The current state-of-the-art method, to isolate and maintain GSC cultures that faithfully mimic the primary tumor, is to use serum-free (SF) media conditions developed for neural stem cells (NSCs). Here we have investigated the outcome of explanting 218 consecutively collected GBM patient samples under both SF and standard, serum-containing media conditions. The frequency of maintainable SF cultures (SFCs) was most successful, but for a subgroup of GBM specimens, a viable culture could only be established in serum-containing media, called exclusive serum culture (ESC). ESCs expressed nestin and SOX2, and displayed all functional characteristics of a GSC, that is, extended proliferation, sustained self-renewal and orthotopic tumor initiation. Once adapted to the in vitro milieu they were also sustainable in SF media. Molecular analyses showed that ESCs formed a discrete group that was most related to the mesenchymal GBM subtype. This distinct subgroup of GBM that would have evaded modeling in SF conditions only provide unique cell models of GBM inter-tumor heterogeneity.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Madre Neoplásicas/patología , Células-Madre Neurales/patología , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Proliferación Celular/fisiología , Ratones TransgénicosRESUMEN
Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient-derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5high ), there were two phenotypically distinct groups: one that was dependent on LGR5 for its malignant properties and another that was unaffected by changes in LGR5 expression. The LGR5-responding cultures could be identified by their significantly higher self-renewal capacity as measured by extreme limiting dilution assay (ELDA), and these LGR5high -ELDAhigh cultures were also significantly more malignant and invasive compared to the LGR5high -ELDAlow cultures. This showed that LGR5 expression alone would not be a strict marker of LGR5 responsiveness. In a search for additional biomarkers, we identified LPAR4, CCND2, and OLIG2 that were significantly upregulated in LGR5-responsive GSC cultures, and we found that OLIG2 together with LGR5 were predictive of GSC radiation and drug response. Overall, we show that LGR5 regulates the malignant phenotype in a subset of patient-derived GSC cultures, which supports its potential as a predictive GBM biomarker. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Movimiento Celular , Proliferación Celular , Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Autorrenovación de las Células , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/efectos de la radiación , Factor de Transcripción 2 de los Oligodendrocitos/genética , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Fenotipo , Tolerancia a Radiación , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Células Tumorales CultivadasRESUMEN
The loss of the tumour suppressor E-cadherin (Cdh1) is a key event during tumourigenesis and epithelial-mesenchymal transition (EMT). Transforming growth factor-ß (TGFß) triggers EMT by inducing the expression of non-histone chromatin protein High Mobility Group A2 (HMGA2). We have previously shown that HMGA2, together with Smads, regulate a network of EMT-transcription factors (EMT-TFs) like Snail1, Snail2, ZEB1, ZEB2 and Twist1, most of which are well-known repressors of the Cdh1 gene. In this study, we show that the Cdh1 promoter is hypermethylated and epigenetically silenced in our constitutive EMT cell model, whereby HMGA2 is ectopically expressed in mammary epithelial NMuMG cells and these cells are highly motile and invasive. Furthermore, HMGA2 remodels the chromatin to favour binding of de novo DNA methyltransferase 3A (DNMT3A) to the Cdh1 promoter. E-cadherin expression could be restored after treatment with the DNA de-methylating agent 5-aza-2'-deoxycytidine. Here, we describe a new epigenetic role for HMGA2, which follows the actions that HMGA2 initiates via the EMT-TFs, thus achieving sustained silencing of E-cadherin expression and promoting tumour cell invasion.
Asunto(s)
Cadherinas/genética , Transición Epitelial-Mesenquimal/genética , Silenciador del Gen , Proteína HMGA2/metabolismo , Antígenos CD , Azacitidina/análogos & derivados , Azacitidina/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Factor de Unión a CCCTC , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Metiltransferasa 3A , Decitabina , Femenino , Humanos , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Deciphering molecular mechanisms that control epithelial-to-mesenchymal transition (EMT) contributes to our understanding of how tumor cells become invasive and competent for intravasation. We have established that transforming growth factor ß activates Smad proteins, which induce expression of the embryonic factor high mobility group A2 (HMGA2), which causes mesenchymal transition. HMGA2 associates with Smad complexes and induces expression of an established regulator of EMT, the zinc finger transcription factor Snail. We now show that HMGA2 can also induce expression of a second regulator of EMT, the basic helix-loop-helix transcription factor Twist. Silencing of endogenous Twist demonstrated that this protein acts in a partially redundant manner together with Snail. Double silencing of Snail and Twist reverts mesenchymal HMGA2-expressing cells to a more epithelial phenotype when compared with single silencing of Snail or Twist. Furthermore, HMGA2 can directly associate with A:T-rich sequences and promote transcription from the Twist promoter. The new evidence proposes a model whereby HMGA2 directly induces multiple transcriptional regulators of the EMT program and, thus, is a potential biomarker for carcinomas displaying EMT during progression to more advanced stages of malignancy.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Transición Epitelial-Mesenquimal , Proteína HMGA2/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/biosíntesis , Elementos de Respuesta , Proteína 1 Relacionada con Twist/biosíntesis , Animales , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Proteína HMGA2/genética , Células Hep G2 , Humanos , Ratones , Modelos Biológicos , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Proteínas Nucleares/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/genéticaRESUMEN
Epithelial plasticity characterizes embryonic development and diseases such as cancer. Epithelial-mesenchymal transition (EMT) is a reversible and guided process of plasticity whereby embryonic or adult epithelia acquire mesenchymal properties. Multiple signaling pathways control EMT, and the transforming growth factor ß (TGFß) pathway plays a central role as its inducer. Here, we analyzed the role of the tumor suppressor protein p53 in TGFß-induced EMT in a well-established mammary epithelial cell model. We found that diploid NMuMG mammary cells bi-allelically express a wild type and a missense mutant (R277C) form of p53. Global reduction of both forms of p53 led to an enhanced EMT response to TGFß. Conversely, stabilization of wild type p53 using the compound nutlin had a negative impact on EMT. After silencing both p53 forms, rescue experiments using either wild type or R277C mutant p53 revealed that wild type p53 inhibited, whereas the R277C mutant did not significantly affect, the TGFß-driven EMT response. Under serum-free culture conditions, silencing of total p53 levels led to higher numbers of mammospheres characterized by larger size. Rescue of the silenced endogenous p53 with R277C mutant p53, in contrast, suppressed both size and numbers of the mammospheres. This work proposes that wild type p53 controls the efficiency by which mammary epithelial cells undergo EMT in response to TGFß.
Asunto(s)
Células Epiteliales/citología , Transición Epitelial-Mesenquimal/genética , Glándulas Mamarias Animales/citología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Células Cultivadas , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Ratones , MutaciónRESUMEN
There is ample support for developmental regulation of glioblastoma stem cells. To examine how cell lineage controls glioblastoma stem cell function, we present a cross-species epigenome analysis of mouse and human glioblastoma stem cells. We analyze and compare the chromatin-accessibility landscape of nine mouse glioblastoma stem cell cultures of three defined origins and 60 patient-derived glioblastoma stem cell cultures by assay for transposase-accessible chromatin using sequencing. This separates the mouse cultures according to cell of origin and identifies three human glioblastoma stem cell clusters that show overlapping characteristics with each of the mouse groups, and a distribution along an axis of proneural to mesenchymal phenotypes. The epigenetic-based human glioblastoma stem cell clusters display distinct functional properties and can separate patient survival. Cross-species analyses reveals conserved epigenetic regulation of mouse and human glioblastoma stem cells. We conclude that epigenetic control of glioblastoma stem cells primarily is dictated by developmental origin which impacts clinically relevant glioblastoma stem cell properties and patient survival.
Asunto(s)
Glioblastoma , Linaje de la Célula/genética , Cromatina/genética , Epigénesis Genética , Glioblastoma/genética , Humanos , Células Madre NeoplásicasRESUMEN
Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor ß (TGFß) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGFß1 signaling activity. Exogenous TGFß1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGFß pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Perfilación de la Expresión Génica/métodos , Glioblastoma/metabolismo , Células Madre Neoplásicas/citología , Transducción de Señal , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Neoplasias Encefálicas/genética , Diferenciación Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Humanos , Ratones , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Factores de Transcripción de la Familia Snail/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The Rho GTPases are involved in many signaling pathways and cellular functions, including the organization of the actin cytoskeleton, regulation of transcription, cell motility, and cell division. The p21 (Cdc42/Rac)-activated kinase PAK mediates a number of biological effects downstream of these Rho GTPases (reviewed by [1]). The phosphorylation state of mammalian PAK is highly regulated: upon binding of GTPases, PAK is potently activated by autophosphorylation at multiple sites, although the mechanisms of PAK downregulation are not known. We now report two PP2C-like serine/threonine phosphatases (POPX1 and POPX2) that efficiently inactivate PAK. POPX1 was isolated as a binding partner for the PAK interacting guanine nucleotide exchange factor PIX. The dephosphorylating activity of POPX correlates with an ability to block the in vivo effects of active PAK. Consonant with these effects on PAK, POPX can also inhibit actin stress fiber breakdown and morphological changes driven by active Cdc42(V12). The association of the POPX phosphatases with PAK complexes may allow PAK to cycle rapidly between active and inactive states; it represents a unique regulatory component of the signaling pathways of the PAK kinase family.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Células COS , Proteínas de Ciclo Celular/metabolismo , Regulación hacia Abajo , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Fenotipo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosforilación , Unión Proteica , Proteína Fosfatasa 2 , Proteína Fosfatasa 2C , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 ActivadasRESUMEN
Epithelial-mesenchymal transition (EMT) refers to plastic changes in epithelial tissue architecture. Breast cancer stromal cells provide secreted molecules, such as transforming growth factor ß (TGFß), that promote EMT on tumor cells to facilitate breast cancer cell invasion, stemness and metastasis. TGFß signaling is considered to be abnormal in the context of cancer development; however, TGFß acting on breast cancer EMT resembles physiological signaling during embryonic development, when EMT generates or patterns new tissues. Interestingly, while EMT promotes metastatic fate, successful metastatic colonization seems to require the inverse process of mesenchymal-epithelial transition (MET). EMT and MET are interconnected in a time-dependent and tissue context-dependent manner and are coordinated by TGFß, other extracellular proteins, intracellular signaling cascades, non-coding RNAs and chromatin-based molecular alterations. Research on breast cancer EMT/MET aims at delivering biomolecules that can be used diagnostically in cancer pathology and possibly provide ideas for how to improve breast cancer therapy.
Asunto(s)
Neoplasias de la Mama/metabolismo , Reprogramación Celular , Transición Epitelial-Mesenquimal , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Epigénesis Genética , Femenino , Humanos , Ratones , Células Madre Neoplásicas/metabolismo , ARN no Traducido/metabolismoRESUMEN
Reactive oxygen species (ROS), the most prominent free radicals produced in cells, can have both beneficial and detrimental effects on them. Many genes are known to be involved in ROS regulation. P53 inducible gene 3 (PIG3 or TP53I3) was identified in an analysis of genes induced by p53 before the onset of apoptosis. It is a widely conserved gene between many species. Until now it has been shown to exert two disparate cellular roles. The first is that of ROS producer linked to p53 induced apoptosis. In this context, it exhibits a NADPH dependent reductase activity with orthoquinones. The second is that of a component of the DNA damage response pathway. While it is considered as a p53 dependent pro-apoptotic gene, it is rarely affected in cancer. This data does not support an anti-tumor activity. In the present review we present and discuss aspects on the regulation and function of this factor and how it is implicated in cancer. We conclude by proposing that PIG3 may possibly have a role in cancer cell survival.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Daño del ADN , Reparación del ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias/metabolismo , Estrés Oxidativo , Proteínas Proto-Oncogénicas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Supervivencia Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias/genética , Neoplasias/patología , Proteínas Proto-Oncogénicas/genética , Transducción de SeñalRESUMEN
Rho GTPases and their downstream effectors regulate changes in the actin cytoskeleton that underlie cell motility and adhesion. They also participate, with RhoA, in the regulation of gene transcription by activating serum response factor (SRF)-mediated transcription from the serum response element (SRE). SRF-mediated transcription is also promoted by several proteins that regulate the polymerization or stability of actin. We have previously identified a family of PP2C phosphatases, POPXs, which can dephosphorylate the CDC42/RAC-activated kinase PAK and downregulate its enzymatic and actin cytoskeletal activity. We now report that POPX2 interacts with the formin protein mDia1 (DIAPH1). This interaction is enhanced when mDia1 is activated by RhoA. The binding of POPX2 to mDia1 or to an mDia-containing complex greatly decreases the ability of mDia1 to activate transcription from the SRE. We propose that the interaction between mDia1 and POPX2 (PPM1F) serves to regulate both the actin cytoskeleton and SRF-mediated transcription, and to link the CDC42/RAC1 pathways with those of RhoA.
Asunto(s)
Proteínas Portadoras/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Transducción de Señal , Proteína de Unión al GTP rhoA/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/genética , Forminas , Inmunoprecipitación , Ratones , Modelos Biológicos , Células 3T3 NIH , Fosfoproteínas Fosfatasas/genética , Unión Proteica , ARN Interferente Pequeño/genética , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Fibras de Estrés/metabolismo , Transcripción GenéticaRESUMEN
Epithelial-mesenchymal transition (EMT) is important during embryonic cell layer movement and tumor cell invasiveness. EMT converts adherent epithelial cells to motile mesenchymal cells, favoring metastasis in the context of cancer progression. Transforming growth factor-beta (TGF-beta) triggers EMT via intracellular Smad transducers and other signaling proteins. We previously reported that the high mobility group A2 (HMGA2) gene is required for TGF-beta to elicit EMT in mammary epithelial cells. In the present study we investigated the molecular mechanisms by which HMGA2 induces EMT. We found that HMGA2 regulates expression of many important repressors of E-cadherin. Among these, we analyzed in detail the zinc-finger transcription factor SNAIL1, which plays key roles in tumor progression and EMT. We demonstrate that HMGA2 directly binds to the SNAIL1 promoter and acts as a transcriptional regulator of SNAIL1 expression. Furthermore, we observed that HMGA2 cooperates with the TGF-beta/Smad pathway in regulating SNAIL1 gene expression. The mechanism behind this cooperation involves physical interaction between these factors, leading to an increased binding of Smads to the SNAIL1 promoter. SNAIL1 seems to play the role of a master effector downstream of HMGA2 for induction of EMT, as SNAIL1 knock-down partially reverts HMGA2-induced loss of epithelial differentiation. The data propose that HMGA2 acts in a gene-specific manner to orchestrate the transcriptional network necessary for the EMT program.