scholarly journals P11.54 Identification of PDGFRA and MYC(N) as somatic driver genes in Glioblastoma

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii55-iii56
Author(s):  
A D Berezovsky ◽  
A Transou ◽  
S Irtenkauf ◽  
L Poisson ◽  
K Hank Wu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Lines ◽  
Gene Amplification ◽  
Median Survival ◽  
Tyrosine Kinases ◽  
Target Genes ◽  
Expression Patterns ◽  
Mycn Amplification ◽  
Driver Genes

Abstract BACKGROUND Somatic oncogene amplification happens frequently in glioblastoma (GBM). The second most frequently amplified gene encoding receptor tyrosine kinases in GBMs is platelet derived growth factor alpha (PDGFRA) (15%). In contrast, MYC and MYCN amplification occurs in 1.6% and 2.9%, respectively. Our goal was to characterize the role of PDGFRɑ and Myc in GBM. MATERIAL AND METHODS Neurosphere cultures were implanted in cohorts of 10–15 nude mice. 5 PDX lines, presenting median survival of 29–59 days were classified as short survivors, and 5 lines with median survival between 104–134 days classified as long survivors. Total RNA was extracted from PDX terminal tumors (3 biological replicates) and sequenced in a paired-end read format. Mouse reads were filtered out using Xenome. MYC and PDGFRA expression patterns were analyzed in tissue microarrays representing duplicated samples from 40 glioma neurosphere-derived PDX lines by IHC (1 anaplastic oligodendroglioma, 8 recurrent GBM with 2 newly diagnosed/recurrent pairs). Normalized staining intensity (MI) and area (A) were quantified using Fiji/ImageJ. RESULTS PDGFRA, MYC, MYCN gene amplifications were represented in a molecularly diverse panel of GBM patient-derived cancer stem-like cells (CSC) and orthotopic mouse xenografts (PDX). Transforming to a normal distribution (log10), 4/13 of cell lines had a PDGFRA mRNA expression (RPKM) higher than 1.5. Similarly, one PDX line had a staining index of greater than 10, 11 (27.5%) had an index between 5–10. The range of intra-tumoral variance, represented by standard deviation, was 0.09–24.25 highlighting the heterogeneity of PDGFRɑ expression. PDGFRɑ phosphorylation (Y754) did not differ between 8 cell lines cultured in NMGF, but deviated in alternate medias without growth factors, supplemented with FBS. In comparison, MYC(N) mRNA expression is only elevated in the context of a known amplification. Furthermore, a a MYC activity signature consisting of 18 target genes was only evident in the 5 amplified CSC lines. Taking advantage of genomic heterogeneity, we have isolated subclones lacking PDGFRA amplification from a PDGFRA amplified GBM CSC. The absence of PDGFRA amplification reduced the self-renewal potential to 37% of the PDGFRA amplified cell population (p=0.001) in clone 1 and 57% in clone 2 (p=0.013). Pertaining to determinants of in vivo survival, MYC was altered in 80% of short survivors (2/5 MYC, 2/5 MYCN amplification) and in 0% of long survivors. Myc signature was highly correlated with in vivo survival (Pearsons’ corr. = -0.77) and MYC gene expression was correlated with in vivo TMZ resistance (corr. = 0.7). CONCLUSION These results suggest that PDGFRɑ expression and activity can occur in the absence of gene amplification, while Myc activity is dependent on gene amplification. Both oncogenes drive oncogenic pathways that should be explored as therapeutic targets.

scholarly journals Id1 is a common downstream target of oncogenic tyrosine kinases in leukemic cells

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1981-1992 ◽  
Author(s):  
Winnie F. Tam ◽  
Ting-Lei Gu ◽  
Jing Chen ◽  
Benjamin H. Lee ◽  
Lars Bullinger ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tyrosine Kinases ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Hematopoietic Malignancy ◽  
Downstream Target ◽  
Models Of Disease ◽  
Induced Apoptosis

Abstract Oncogenic tyrosine kinases, such as BCR-ABL, TEL-ABL, TEL-PDGFβR, and FLT3-ITD, play a major role in the development of hematopoietic malignancy. They activate many of the same signal transduction pathways. To identify the critical target genes required for transformation in hematopoietic cells, we used a comparative gene expression strategy in which selective small molecules were applied to 32Dcl3 cells that had been transformed to factor-independent growth by these respective oncogenic alleles. We identified inhibitor of DNA binding 1 (Id1), a gene involved in development, cell cycle, and tumorigenesis, as a common target of these oncogenic kinases. These findings were prospectively confirmed in cell lines and primary bone marrow cells engineered to express the respective tyrosine kinase alleles and were also confirmed in vivo in murine models of disease. Moreover, human AML cell lines Molm-14 and K562, which express the FLT3-ITD and BCR-ABL tyrosine kinases, respectively, showed high levels of Id1 expression. Antisense and siRNA based knockdown of Id1-inhibited growth of these cells associated with increased p27Kip1 expression and increased sensitivity to Trail-induced apoptosis. These findings indicate that Id1 is an important target of constitutively activated tyrosine kinases and may be a therapeutic target for leukemias associated with oncogenic tyrosine kinases.

Genes Regulated by NPM-ALK Fusion Kinase Play a Key Role in the Activation of AP-1 Transcription Factors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 245-245 ◽  
Author(s):  
Philipp B. Staber ◽  
Werner Linkesch ◽  
Silvia Schauer ◽  
Gerit Moser ◽  
Marshall E. Kadin ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Lines ◽  
Target Genes ◽  
Expression Patterns ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Anaplastic Lymphoma ◽  
Cancer Pathogenesis ◽  
293 Cells ◽  
Alk Positive

Abstract Background: About half of nodal anaplastic large cell lymphomas (ALCL) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is the product of a t(2;5)(p23;q35) chromosomal translocation. Expression of this protein has been shown to result in neoplastic change. Combining suppression subtractive hybridization (SSH) and cDNA microarray analysis we aimed at elucidating the consequences of NPM-ALK expression. Methods: SSH cDNA libraries were constructed using mRNA from human embryonic kidney (293) cells transfected with active or kinase-dead NPM-ALK constructs, as well as pools of NPM-ALK positive and negative ALCL cell lines. The resulting cDNA clones and genes relevant for cancer pathogenesis were spotted, generating specific cDNA microarrays comprising 4992 genes. mRNA expression patterns were analyzed in individual cell lines. Real time quantitative RT-PCR of 20 selected genes validated the mRNA expression data of the microarrays. Results: Expression of a set of 102 genes distinguishes NPM-ALK-negative (FE-PD, MAC-2A) from NPM-ALK-positive ALCL cell lines (SU-DHL-1, JB-6, SUP-M2, SR-786, DEL and Karpas 299). The majority are involved in regulation of cell cycle and apoptosis. 38 of these genes also discriminate 293 cells with respect to their NPM-ALK expression. Interestingly, AP-1 target genes, such as GM-CSFRA, GM-CSFRB, ARF5, FAS, FASL, and BCL3, are increased in NPM-ALK expressing cell lines. Electrophoretic mobility shift assay (EMSA) verifies NPM-ALK dependent AP-1 DNA binding activity. Conclusion: This study reveals genes specifically regulated by NPM-ALK lymphoma kinase. Further, we demonstrate that AP-1 activation is a critical target of NPM-ALK signalling.

scholarly journals Circular RNA circSDHC serves as a sponge for miR-127-3p to promote the proliferation and metastasis of renal cell carcinoma via the CDKN3/E2F1 axis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Junjie Cen ◽  
Yanping Liang ◽  
Yong Huang ◽  
Yihui Pan ◽  
Guannan Shu ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Target Genes ◽  
Expression Patterns ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Proliferation And Metastasis

Abstract Background There is increasing evidence that circular RNAs (circRNAs) have significant regulatory roles in cancer development and progression; however, the expression patterns and biological functions of circRNAs in renal cell carcinoma (RCC) remain largely elusive. Method Bioinformatics methods were applied to screen for circRNAs differentially expressed in RCC. Analysis of online circRNAs microarray datasets and our own patient cohort indicated that circSDHC (hsa_circ_0015004) had a potential oncogenic role in RCC. Subsequently, circSDHC expression was measured in RCC tissues and cell lines by qPCR assay, and the prognostic value of circSDHC evaluated. Further, a series of functional in vitro and in vivo experiments were conducted to assess the effects of circSDHC on RCC proliferation and metastasis. RNA pull-down assay, luciferase reporter and fluorescent in situ hybridization assays were used to confirm the interactions between circSDHC, miR-127-3p and its target genes. Results Clinically, high circSDHC expression was correlated with advanced TNM stage and poor survival in patients with RCC. Further, circSDHC promoted tumor cell proliferation and invasion, both in vivo and in vitro. Analysis of the mechanism underlying the effects of circSDHC in RCC demonstrated that it binds competitively to miR-127-3p and prevents its suppression of a downstream gene, CDKN3, and the E2F1 pathway, thereby leading to RCC malignant progression. Furthermore, knockdown of circSDHC caused decreased CDKN3 expression and E2F1 pathway inhibition, which could be rescued by treatment with an miR-127-3p inhibitor. Conclusion Our data indicates, for the first time, an essential role for the circSDHC/miR-127-3p/CDKN3/E2F1 axis in RCC progression. Thus, circSDHC has potential to be a new therapeutic target in patients with RCC.

scholarly journals The In Vivo Selection Method in Breast Cancer Metastasis

2021 ◽  
Vol 22 (4) ◽  
pp. 1886
Author(s):  
Jun Nakayama ◽  
Yuxuan Han ◽  
Yuka Kuroiwa ◽  
Kazushi Azuma ◽  
Yusuke Yamamoto ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Metastasis ◽  
Metastatic Cancer ◽  
Expression Patterns ◽  
Selection Method ◽  
Gene Signatures ◽  
In Vivo Selection ◽  
Metastatic Cancer Cells

Metastasis is a complex event in cancer progression and causes most deaths from cancer. Repeated transplantation of metastatic cancer cells derived from transplanted murine organs can be used to select the population of highly metastatic cancer cells; this method is called as in vivo selection. The in vivo selection method and highly metastatic cancer cell lines have contributed to reveal the molecular mechanisms of cancer metastasis. Here, we present an overview of the methodology for the in vivo selection method. Recent comparative analysis of the transplantation methods for metastasis have revealed the divergence of metastasis gene signatures. Even cancer cells that metastasize to the same organ show various metastatic cascades and gene expression patterns by changing the transplantation method for the in vivo selection. These findings suggest that the selection of metastasis models for the study of metastasis gene signatures has the potential to influence research results. The study of novel gene signatures that are identified from novel highly metastatic cell lines and patient-derived xenografts (PDXs) will be helpful for understanding the novel mechanisms of metastasis.

scholarly journals Birdsong Decreases Protein Levels of FoxP2, a Molecule Required for Human Speech

2008 ◽  
Vol 100 (4) ◽  
pp. 2015-2025 ◽  
Author(s):  
Julie E. Miller ◽  
Elizabeth Spiteri ◽  
Michael C. Condro ◽  
Ryan T. Dosumu-Johnson ◽  
Daniel H. Geschwind ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Target Genes ◽  
Expression Patterns ◽  
Vocal Learning ◽  
Brain Regions ◽  
Motor Deficits ◽  
Mrna Levels ◽  
Adult Males ◽  
Protein Levels ◽  
Area X

Cognitive and motor deficits associated with language and speech are seen in humans harboring FOXP2 mutations. The neural bases for FOXP2 mutation-related deficits are thought to reside in structural abnormalities distributed across systems important for language and motor learning including the cerebral cortex, basal ganglia, and cerebellum. In these brain regions, our prior research showed that FoxP2 mRNA expression patterns are strikingly similar between developing humans and songbirds. Within the songbird brain, this pattern persists throughout life and includes the striatal subregion, Area X, that is dedicated to song development and maintenance. The persistent mRNA expression suggests a role for FoxP2 that extends beyond the formation of vocal learning circuits to their ongoing use. Because FoxP2 is a transcription factor, a role in shaping circuits likely depends on FoxP2 protein levels which might not always parallel mRNA levels. Indeed our current study shows that FoxP2 protein, like its mRNA, is acutely downregulated in mature Area X when adult males sing with some differences. Total corticosterone levels associated with the different behavioral contexts did not vary, indicating that differences in FoxP2 levels are not likely attributable to stress. Our data, together with recent reports on FoxP2's target genes, suggest that lowered FoxP2 levels may allow for expression of genes important for circuit modification and thus vocal variability.

A role for the EphA family in the topographic targeting of vomeronasal axons

Development ◽  
2001 ◽  
Vol 128 (6) ◽  
pp. 895-906
Author(s):  
B. Knoll ◽  
K. Zarbalis ◽  
W. Wurst ◽  
U. Drescher
Keyword(s):  
Tyrosine Kinases ◽  
Expression Patterns ◽  
Eph Receptors ◽  
Accessory Olfactory Bulb ◽  
In Vivo Experiments ◽  
Receptor Concentration ◽  
High Ligand

We have investigated the role of the Eph family of receptor tyrosine kinases and their ligands in the establishment of the vomeronasal projection in the mouse. Our data show intriguing differential expression patterns of ephrin-A5 on vomeronasal axons and of EphA6 in the accessory olfactory bulb (AOB), such that axons with high ligand concentration project onto regions of the AOB with high receptor concentration and vice versa. These data suggest a mechanism for development of this projection that is the opposite of the repellent interaction between Eph receptors and ligands observed in other systems. In support of this idea, when given the choice of whether to grow on lanes containing EphA-F(c)/laminin or F(c)/laminin protein (in the stripe assay), vomeronasal axons prefer to grow on EphA-F(c)/laminin. Analysis of ephrin-A5 mutant mice revealed a disturbance of the topographic targeting of vomeronasal axons to the AOB. In summary, these data, which are derived from in vitro and in vivo experiments, indicate an important role of the EphA family in setting up the vomeronasal projection.

In vitro and in vivo culture effects on mRNA expression of genes involved in metabolism and apoptosis in bovine embryos

2005 ◽  
Vol 17 (8) ◽  
pp. 775 ◽  
Author(s):  
Hiemke M. Knijn ◽  
Christine Wrenzycki ◽  
Peter J. M. Hendriksen ◽  
Peter L. A. M. Vos ◽  
Elly C. Zeinstra ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Mrna Expression ◽  
Critical Period ◽  
Glucose Transporter ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Glut 4 ◽  
Gene Transcripts

Bovine blastocysts produced in vitro differ substantially from their in vivo-derived counterparts with regard to glucose metabolism, level of apoptosis and mRNA expression patterns. Maternal embryonic genomic transition is a critical period in which these changes could be induced. The goals of the present study were twofold: (1) to identify the critical period of culture during which the differences in expression of gene transcripts involved in glucose metabolism are induced; and (2) to identify gene transcripts involved in apoptosis that are differentially expressed in in vitro- and in vivo-produced blastocysts. Relative abundances of transcripts for the glucose transporters Glut-1, Glut-3, Glut-4 and Glut-8, and transcripts involved in the apoptotic cascade, including BAX, BCL-XL, XIAP and HSP 70.1, were analysed by a semiquantitative reverse transcription–polymerase chain reaction assay in single blastocysts produced in vitro or in vivo for specific time intervals, that is, before or after maternal embryonic transition. Whether the culture environment was in vitro or in vivo affected the expression of glucose transporter transcripts Glut-3, Glut-4 and Glut-8. However, the critical period during culture responsible for these changes, before or after maternal embryonic transition, could not be determined. With the exception of XIAP, no effects of culture system on the mRNA expression patterns of BAX, BCL-XL and HSP 70.1 could be observed. These data show that expression of XIAP transcripts in expanded blastocysts is affected by in vitro culture. These findings add to the list of bovine genes aberrantly expressed in culture conditions, but do not support the hypothesis that maternal embryonic transition is critical in inducing the aberrations in gene expression patterns studied here.

121 DIFFERENTIAL mRNA EXPRESSION BETWEEN IN VIVO AND IN VITRO-DERIVED BOS INDICUS AND BOS TAURUS EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 160
Author(s):  
L. Nasser ◽  
P. Stranieri ◽  
A. Gutiérrez-Adán ◽  
M. Clemente ◽  
L. Jorge de Souza ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Repeated Measures ◽  
Bos Taurus ◽  
Receptor Gene ◽  
Expression Patterns ◽  
Bos Indicus ◽  
Growth Factor Receptor ◽  
P53 Gene

Brazil is a leading country in the world of commercial use of in vitro-produced bovine embryos with 200 000 transfers per year. The majority of in vitro-produced embryos are pure breed Nelore and are transferred fresh with 40% pregnancy rate. However, pregnancies are drastically reduced with frozen in vitro embryos. This experiment is part of our effort to learn more about molecular composition and morphology of in vitro-derived embryos that may be responsible for such discrepancy. We examined molecular expression of mRNA transcripts of 6 selected genes; apoptosis Bax,TP53(p53), SHC1SHC(p66), insulin growth factor receptor (IGF2R), stabilization of the plasma membrane PLAC8 and glucose conversion H6PD in in-vivo (control) and in-vitro Nelore and Bos taurus embryos. In vivo embryos were collected from superovulated cows at Day 7. In vitro embryo was produced from oocytes aspirated from live cows. A total of 284 oocytes (4 replicates) were matured and fertilized by standard IVF procedures. Presumptive zygotes were cultured in CR2 medium with 5% BSA in 50 μL drops (25 zygotes per drop) at 39°C under paraffin oil and 5% CO2 in humidified air. Embryos that developed on Days 7 to blastocyst were transferred to recipients, and 10 blastocysts from each replicate were frozen for evaluation of gene expression patterns. Poly(A) mRNA was prepared from 3 groups of pools of 10 in vitro embryos and 10 of control in vivo-derived embryos. The quantification of all gene transcripts was carried out by real-time quantitative RT-PCR using the comparative CT method. Data on mRNA expression were normalized to the endogenous H2a.z and was analyzed by one-way repeated-measures ANOVA. The cleavage rates at Day 2 and number of blastocysts developed at Day 7 were 80.3 ± 3.2 and 42.2 ± 6.4, respectively. The level of expression of IGF2R was significantly (P < 0.05) higher in in vivo-derived embryos than in both groups of in vitro embryos. The expression of all 3 apoptosis genes were lower (P < 0.05) in in vivo than in vitro embryos with exception of p53 gene that was not different between Nelore in vitro and in vivo embryos but was significantly higher (P < 0.05) in Bos taurus in vitro embryos. There was no difference in expression of PLAC8 gene among any tested group of embryos and in expression of H6PD gene between Nelore in vitro and in vivo embryos. We concluded that significant differences in molecular makeup between in vitro and in vivo-derived Nelore embryos exist. Of particular importance seems to be pattern of expression of IGF2R receptor gene known as a good indicator of embryo quality, which promotes proliferation and differentiation. Similarly, higher expression of 2 BAX and p66 genes of apoptosis in in vitro embryos seems to be a further indication of inferior quality of Nelore in vitro-derived embryos that showed to be more profound in Bos taurus in vitro-derived embryos.

Cloning and Characterization of EphA3 (Hek) Gene Promoter: DNA Methylation Regulates Expression in Hematopoietic Tumor Cells

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2477-2486 ◽  
Author(s):  
Mirella Dottori ◽  
Michelle Down ◽  
Andreas Hüttmann ◽  
David R. Fitzpatrick ◽  
Andrew W. Boyd
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Expression Patterns ◽  
Gene Promoter ◽  
Restriction Enzymes ◽  
Clinical Samples ◽  
Cpg Dinucleotides ◽  
Normal Tissues

The Eph family of receptor tyrosine kinases (RTK) has restricted temporal and spatial expression patterns during development, and several members are also found to be upregulated in tumors. Very little is known of the promoter elements or regulatory factors required for expression of Eph RTK genes. In this report we describe the identification and characterization of the EphA3 gene promoter region. A region of 86 bp located at −348 bp to −262 bp upstream from the transcription start site was identified as the basal promoter. This region was shown to be active in both EphA3-expressing and -nonexpressing cell lines, contrasting with the widely different levels of EphA3 expression. We noted a region rich in CpG dinucleotides downstream of the basal promoter. Using Southern blot analyses with methylation-sensitive restriction enzymes and bisulfite sequencing of genomic DNA, sites of DNA methylation were identified in hematopoietic cell lines which correlated with their levels of EphA3 gene expression. We showed that EphA3 was not methylated in normal tissues but that a subset of clinical samples from leukemia patients showed extensive methylation, similar to that observed in cell lines. These results suggest that DNA methylation may be an important mechanism regulating EphA3 transcription in hematopoietic tumors.

Tyrosine kinase profiling guides combined therapeutic strategies in head and neck cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 6078-6078
Author(s):  
R. Rodríguez-Barrueco ◽  
M. Ortíz-Ruiz ◽  
J. J. Cruz ◽  
A. Ocana ◽  
A. Pandiella
Keyword(s):  
Head And Neck Cancer ◽  
Head And Neck ◽  
Cell Lines ◽  
Neck Cancer ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Drug Combinations ◽  
Metastatic Setting

6078 Background: Squamous cell carcinoma of the head and neck (SCCHN) is still an incurable disease in the metastatic setting. A particular subgroup of proteins implicated in the head and neck cancer are the tyrosine kinases (TK). Therapeutic inhibition of several of them including the EGF receptor with cetuximab in combination with radiotherapy or chemotherapy has shown to be clinically useful. Beyond EGFR, oncogenic activation of other TKs may be implicated in the genesis/progression of SCCHN. In this context, the identification of the TKs activated in SCCHN is a must in order to adequately target these kinases with already available inhibitors. Methods: Here we have investigated activated tyrosine kinases in head and neck cancer tumors derived from patients using a human phospho protein array for 42 receptor tyrosine kinases (RTK). Western-blot experiments were performed to validate each phospho RTK in tumors from patients. The same approach was followed in a series of head and neck cancer cell lines. In vivo xenografted models were used to study the antiproliferative effect of the combination of specific TK inhibitors against them. Results: TK receptors of the EGF and the VEGF family were the mostly activated in tumors derived from patients. 90% of patients revealed high pEGFR content. In addition, other EGFR/HER family receptors, such as HER3, were also activated (phosphorylated) in samples from patients. These data were corroborated in the SCCHN cell lines. In these cells, other RTK signalling intermediates were also active. Particularly, the Akt and FAK kinases. Combination of the anti-EGFR-HER2 TK inhibitor lapatinib with dasatinib (that targets FAK) was synergistic in vitro. Combination of lapatinib with the anti-VEGFR TK inhibitor pazopanib was inefficient in vitro, but resulted in a better trend in response in the in vivo xenografted models, as compared to the action of the single agents. Conclusions: Rational target drug combinations should be based on the identification of activated TK receptors or downstream signalling molecules. In head and neck cancer combination strategies using anti-EGFR/HER, anti-FAK, and anti-VEGFR compounds increases the action of individual treatments. These results open the door for future clinical development of these drug combinations. No significant financial relationships to disclose.

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