Transcription profiling by array of human PTEN null U87MG glioblastoma cells transfected with wild-type PTEN, mutant PTEN or GFP controls to investigate the effects of PTEN activity on gene expression
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Schurch Nicholas
PROVIDER: S-ECPF-MTAB-989 | biostudies-other |
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Ferroportin [FPN; Slc40a1 (solute carrier family 40, member 1)] is a transmembrane iron export protein expressed in macrophages and duodenal enterocytes. Heterozygous mutations in the FPN gene result in an autosomal dominant form of iron overload disorder, type-4 haemochromatosis. FPN mutants either have a normal iron export activity but have lost their ability to bind hepcidin, or are defective in their iron export function. The mutant protein has been suggested to act as a dominant negative over the wt (wild-type) protein by multimer formation. Using transiently transfected human epithelial cell lines expressing mouse FPN modified by the addition of a haemagglutinin or c-Myc epitope at the C-terminus, we show that the wtFPN is found at the plasma membrane and in Rab5-containing endosomes, as are the D157G and Q182H mutants. However, the delV162 mutant is mostly intracellular in HK2 cells (human kidney-2 cells) and partially addressed at the cell surface in HEK-293 cells (human embryonic kidney 293 cells). In both cell types, it is partially associated with the endoplasmic reticulum and with Rab5-positive vesicles. However, this mutant is complex-glycosylated like the wt protein. D157G and G323V mutants have a defective iron export capacity as judged by their inability to deplete the intracellular ferritin content, whereas Q182H and delV162 have normal iron export function and probably have lost their capacity to bind hepcidin. In co-transfection experiments, the delV162 mutant does not co-localize with the wtFPN, does not prevent its normal targeting to the plasma membrane and cannot be immunoprecipitated in the same complex, arguing against the formation of FPN hetero-oligomers.
| S-EPMC1462719 | biostudies-literature
Project description:Receptor tyrosine kinase (RTK) systems, such as hepatocyte growth factor (HGF) and its receptor c-Met, and epidermal growth factor receptor (EGFR), are responsible for the malignant progression of multiple solid tumors. Recent research shows that these RTK systems comodulate overlapping and dynamically adaptable oncogenic downstream signaling pathways. This study investigates how EGFRvIII, a constitutively active EGFR deletion mutant, alters tumor growth and signaling responses to RTK inhibition in PTEN-null/HGF(+)/c-Met(+) glioma xenografts. We show that a neutralizing anti-HGF monoclonal antibody (L2G7) potently inhibits tumor growth and the activation of Akt and mitogen-activated protein kinase (MAPK) in PTEN-null/HGF(+)/c-Met(+)/EGFRvIII(-) U87 glioma xenografts (U87wt). Isogenic EGFRvIII(+) U87 xenografts (U87-EGFRvIII), which grew five times more rapidly than U87-wt xenografts, were unresponsive to EGFRvIII inhibition by erlotinib and were only minimally responsive to anti-HGF monoclonal antibodies. EGFRvIII expression diminished the magnitude of Akt inhibition and completely prevented MAPK inhibition by L2G7. Despite the lack of response to L2G7 or erlotinib as single agents, their combination synergized to produce substantial antitumor effects (inhibited tumor cell proliferation, enhanced apoptosis, arrested tumor growth, prolonged animal survival), against subcutaneous and orthotopic U87-EGFRvIII xenografts. The dramatic response to combining HGF:c-Met and EGFRvIII pathway inhibitors in U87-EGFRvIII xenografts occurred in the absence of Akt and MAPK inhibition. These findings show that combining c-Met and EGFRvIII pathway inhibitors can generate potent antitumor effects in PTEN-null tumors. They also provide insights into how EGFRvIII and c-Met may alter signaling networks and reveal the potential limitations of certain biochemical biomarkers to predict the efficacy of RTK inhibition in genetically diverse cancers.
| S-EPMC2846399 | biostudies-literature
Project description:BackgroundMyocilin is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Gln368stop (Q368X) and Pro370Leu (P370L) have been identified in patients. The exact role of myocilin and its functional association with glaucoma are still unclear. In the present study, we established tetracycline-inducible (Tet-on) wild type and mutant myocilin-green fluorescence protein (GFP) expressing RGC5 stable cell lines and studied the changes in cell migration and barrier function upon induction.Methodology/principal findingsAfter several rounds of selection, clones that displayed low, moderate, or high expression of wild type, Q368X or P370L myocilin-GFP upon doxycycline (Dox) induction were obtained. The levels of wild type and mutant myocilin-GFP in various clones were confirmed by Western blotting. Compared to non-induced controls, the cell migration was retarded, the actin stress fibers were fewer and shorter, and the trypsinization time needed for cells to round up was reduced when wild type or mutant myocilin was expressed. The barrier function was in addition aberrant following induced expression of wild type, Q368X or P370L myocilin. Immunoblotting further showed that tight junction protein occludin was downregulated in induced cells.Conclusions/significanceTet-on inducible, stable RGC5 cell lines were established. These cell lines, expressing wild type or mutant (Q368X or P370L) myocilin-GFP upon Dox induction, are valuable in facilitating studies such as proteomics, as well as functional and pathogenesis investigations of disease-associated myocilin mutants. The barrier function was found impaired and the migration of cells was hindered with induced expression of wild type and mutant myocilin in RGC5 cell lines. The reduction in barrier function might be related to the declined level of occludin. The retarded cell migration was consistent with demonstrated myocilin phenotypes including the loss of actin stress fibers, lowered RhoA activities and compromised cell-matrix adhesiveness.
| S-EPMC3474840 | biostudies-literature
Project description:TP53 R175H mutation is one of the most common mutations in human cancers and cancer cells with this mutation stably express R175H protein in the nucleus. To identify the synthetic lethal gene interacted with the R175H, we conducted the high throughput screening by using tetracyclin inducible R175H expression system in SF126 cells and comprehensive shRNA library carried by lenti virus. We identified 906 candidate gene suppressions that may lead to accelerated cell growth inhibition under the presence of R175H. Inhibitor of differentiation 1 (ID1) was one of the candidate genes and suppression of ID1 by siRNA resulted in acceleration of growth inhibition of cell lines expressing endogenous R175H but not in TP53 null cell lines. The transient expression of R175H in TP53 null cell lines and suppression of ID1 and/or TP53 R175H in cell lines with endogenous R175H revealed that the cell growth inhibition by ID1 suppression was dependent on R175H expression but not other common p53 mutants (R273H). Flow cytometric (FACS) analysis exhibited that ID1 suppression resulted in G1 arrest and the arrest was accelerated by suppression of R175H. In conclusion, ID1 is a synthetic lethal gene interacted with R175H and is considered to be a novel molecular target of cancer therapy for R175H expressing cells. R175H expression(Tet-on) group was labeled by Cy5, and p53-null(Tet-off) group was labeled by Cy3. Three independent experiments were conducted (We have triplicate data).
| S-ECPF-GEOD-33362 | biostudies-other
Project description:Microglia and macrophages are the largest component of the inflammatory infiltrate in glioblastoma (GBM). However, whether there are differences in their representation and activity in the prognostically-favorable isocitrate dehydrogenase (IDH)-mutated compared to -wild type GBMs is unknown. Studies on human specimens of untreated IDH-mutant GBMs are rare given they comprise 10% of all GBMs and often present at lower grades, receiving treatments prior to dedifferentiation that can drastically alter microglia and macrophage phenotypes. We were able to obtain large samples of four previously untreated IDH-mutant GBM. Using flow cytometry, immunofluorescence techniques with automated segmentation protocols that quantify at the individual-cell level, and comparison between single-cell RNA-sequencing (scRNA-seq) databases of human GBM, we discerned dissimilarities between GBM-associated microglia and macrophages (GAMMs) in IDH-mutant and -wild type GBMs. We found there are significantly fewer GAMM in IDH-mutant GBMs, but they are more pro-inflammatory, suggesting this contributes to the better prognosis of these tumors. Our pro-inflammatory score which combines the expression of inflammatory markers (CD68/HLA-A, -B, -C/TNF/CD163/IL10/TGFB2), Iba1 intensity, and GAMM surface area also indicates that more pro-inflammatory GAMMs are associated with longer overall survival independent of IDH status. Interrogation of scRNA-seq databases demonstrates microglia in IDH-mutants are mainly pro-inflammatory, while anti-inflammatory macrophages that upregulate genes such as FCER1G and TYROBP predominate in IDH-wild type GBM. Taken together, these observations are the first head-to-head comparison of GAMMs in treatment-naïve IDH-mutant versus -wild type GBMs. Our findings highlight biological disparities in the innate immune microenvironment related to IDH prognosis that can be exploited for therapeutic purposes.
| S-EPMC6517100 | biostudies-literature
Project description:Genomic alteration can reshape tumor microenvironment to drive tumor malignancy. However, how PTEN deficiency influences microenvironment-mediated cell-cell interactions in glioblastoma (GBM) remains unclear. Here, we show that PTEN deficiency induces a symbiotic glioma-M2 macrophage interaction to support glioma progression. Mechanistically, PTEN-deficient GBM cells secrete high levels of galectin-9 (Gal-9) via the AKT-GSK3β-IRF1 pathway. The secreted Gal-9 drives macrophage M2 polarization by activating its receptor Tim-3 and downstream pathways in macrophages. These macrophages, in turn, secrete VEGFA to stimulate angiogenesis and support glioma growth. Furthermore, enhanced Gal-9/Tim-3 expression predicts poor outcome in glioma patients. In GBM models, blockade of Gal-9/Tim-3 signaling inhibits macrophage M2 polarization and suppresses tumor growth. Moreover, α-lactose attenuates glioma angiogenesis by down-regulating macrophage-derived VEGFA, providing a novel antivascularization strategy. Therefore, our study suggests that blockade of Gal-9/Tim-3 signaling is effective to impair glioma progression by inhibiting macrophage M2 polarization, specifically for PTEN-null GBM.
| S-EPMC9269888 | biostudies-literature
Project description:35 Melanoma cell lines hybridized to Affymetrix Hu133_Plus 2 microarrays were analysed for genes differentially expressed between cell lines carrying wild-type p14ARF and those with mutant 14ARF. All of these cell lines contained wild-type p53 (so that the effects of p14ARF mutations could be analysed without contamination from p53). Experiment Overall Design: The aim of this study was to identify downstream effectors of p14ARF in melanoma. Relevant paper: Packer et al. (2007) Int J Cancer.
| S-ECPF-GEOD-7152 | biostudies-other
Project description:While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells.
| S-EPMC4315579 | biostudies-literature
Project description:Background & aimsβ-catenin is a well-known effector of the Wnt pathway, and a key player in cadherin-mediated cell adhesion. Oncogenic mutations of β-catenin are very frequent in paediatric liver primary tumours. Those mutations are mostly heterozygous, which allows the co-expression of wild-type (WT) and mutated β-catenins in tumour cells. We investigated the interplay between WT and mutated β-catenins in liver tumour cells, and searched for new actors of the β-catenin pathway.MethodsUsing an RNAi strategy in β-catenin-mutated hepatoblastoma (HB) cells, we dissociated the structural and transcriptional activities of β-catenin, which are carried mainly by WT and mutated proteins, respectively. Their impact was characterised using transcriptomic and functional analyses. We studied mice that develop liver tumours upon activation of β-catenin in hepatocytes (APCKO and β-cateninΔexon3 mice). We used transcriptomic data from mouse and human HB specimens, and used immunohistochemistry to analyse samples.ResultsWe highlighted an antagonistic role of WT and mutated β-catenins with regard to hepatocyte differentiation, as attested by alterations in the expression of hepatocyte markers and the formation of bile canaliculi. We characterised fascin-1 as a transcriptional target of mutated β-catenin involved in tumour cell differentiation. Using mouse models, we found that fascin-1 is highly expressed in undifferentiated tumours. Finally, we found that fascin-1 is a specific marker of primitive cells including embryonal and blastemal cells in human HBs.ConclusionsFascin-1 expression is linked to a loss of differentiation and polarity of hepatocytes. We present fascin-1 as a previously unrecognised factor in the modulation of hepatocyte differentiation associated with β-catenin pathway alteration in the liver, and as a new potential target in HB.Impact and implicationsThe FSCN1 gene, encoding fascin-1, was reported to be a metastasis-related gene in various cancers. Herein, we uncover its expression in poor-prognosis hepatoblastomas, a paediatric liver cancer. We show that fascin-1 expression is driven by the mutated beta-catenin in liver tumour cells. We provide new insights on the impact of fascin-1 expression on tumour cell differentiation. We highlight fascin-1 as a marker of immature cells in mouse and human hepatoblastomas.
| S-EPMC10159820 | biostudies-literature
Project description:We performed expression mouse profiling of prostates of 3 month PTEN f/f and Pten f/f;R26(ERG) mice and assessed the response to 3 days of castration.
2013-04-25 | GSE46360 | GEO