Project description:The gene expression profiles of two groups of triplicate human glioblastoma (GBM) xenografts grown in immunodeficient rats were compared. The first group of xenografts was derived from a patient biopsy with Epidermal Growth Factor Receptor (EGFR) amplification which grows highly invasive and is independent of angiogenesis. The second group was obtained by introducing a dominant-negative EGFR mutant into the tumor cells, leading to a progression of the tumors to an angiogenic phenotype associated with a transition from a proneural to a mesenchymal GBM molecular subtype.
Project description:Although the oncogenic signalings driven by amplification and mutations of EGF receptor (EGFR) gene play a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here we demonstrate that tripartite motif-containing protein 59 (TRIM59), acting as a new downstream effector of EGFR signaling, regulates STAT3 activation in glioblastoma. EGFR signaling leads to TRIM59 upregulation through SOX9 that results in enhancing TRIM59 interaction with STAT3 in nucleus, and inhibiting STAT3 association with TC45 (the nuclear form of T cell protein tyrosine phosphatase TC-PTP), thereby maintaining STAT3 phosphorylation and activation and promoting tumorigenesis. Silencing TRIM59 suppresses cell proliferation, migration, and orthotopic xenograft brain tumorigenesis of GBM cells. Moreover, evaluation of GBM patient samples reveals an association among EGFR activation, TRIM59 expression, STAT3 phosphorylation, and poor prognoses. Our study identifies TRIM59 as a new regulator of oncogenic EGFR-STAT3 signaling and a potential therapeutic target for GBM patients with EGFR activation.
Project description:Transcriptional profiling of COLO 320 xenograft tumor cells comparing control COLO 320 xenograft without co-implanted rat MSCs with COLO 320 xenograft with co-implanted rat MSCs. The latter makes co-implanted MSCs visualization possible by using MSCs labeled by GFP under FACS and single cell microscopy. Two-condition experiment, COLO 320 xenograft without rat MSCs [COLO320 MSC(-)] vs. COLO 320 xenograft with rat MSCs [COLO320 MSC(+)]. Biological replicates: 1 control, 1 sample, paired xengraft tumor cells grown and harvested from the same mouse host. One replicate per array.
Project description:As a first step towards identifying the target genes of EGFR activity in glioma cells, genome-wide expression analyses were performed using the Affymetrix GeneChip Human Genome U133A array. To accomplish this, mRNA expression levels of these genes were measured in the glioblastoma cell lines, U87 and U178, engineered with EGFR by retrovirus transduction (termed U87-EGFR and U178-EGFR respectively), with or without 20 ng/mL EGF treatment for 3 h. U87 and U178 cells engineered to express EGFR were stimulated with or without EGF. The experiment was replicated twice for each U87 and U178 cells.
Project description:Achievement of specific tumor cell targeting remains a challenge for glioma gene therapy. We report here the identification and characterization of a 5’ sequence of human HMGB2 gene for transcriptional targeting to glioblastoma. We performed microarray analysis and found HMGB2 as one of the genes that had a low level of expression in normal human astrocytes, but was significantly up-regulated in glioblastoma cells. Real-time PCR quantification revealed increase in HMBG2 expression level in glioblastoma tissues and cells between 11 to 79 fold over that in normal human brain tissue. With progressive truncation of a 5’-upstream sequence of the HMGB2 gene, we identified a 500-bp fragment that displayed a high transcriptional activity in glioblastoma cells, but a low activity in normal brain cells. Using the sequence to drive the expression of the herpes simplex virus thymidine kinase gene in the context of a baculoviral vector, glioblastoma cells died in the presence of ganciclovir, whereas normal human astrocytes and neurons were not affected. We further confirmed that after intra-tumor injection, the baculoviral vector effectively suppressed the growth of human glioblastoma cells in a mouse xenograft model. Our results suggest that the 5’-upstream sequence of the HMGB2 gene can be used as an efficient, tumor-selective promoter in targeted vectors for glioblastoma gene therapy. U251 cells (n=3) genes level expression were compared to that of normal astrocytes (n=3) to find overexpressed genes in glioblastoma. Highly expressed genes were compared to those found in the litterature. This was selected to clone promoters of highly expressed genes in glioblastomas
Project description:As a first step towards identifying the target genes of EGFR activity in glioma cells, genome-wide expression analyses were performed using the Affymetrix GeneChip Human Genome U133A array. To accomplish this, mRNA expression levels of these genes were measured in the glioblastoma cell lines, U87 and U178, engineered with EGFR by retrovirus transduction (termed U87-EGFR and U178-EGFR respectively), with or without 20 ng/mL EGF treatment for 3 h.
Project description:Background: Glioblastoma mortality is driven by tumour progression or recurrence despite administering a therapeutic arsenal consisting of surgical resection, radiation, and alkylating chemotherapy. The genetic changes underlying tumour progression and chemotherapy resistance are poorly understood. Methods: In this work, we sought to define the relationship between EGFR amplification status, EGFR mRNA expression, and EGFR pathway activity. We compared RNA-sequencing data from matched primary and recurrent tumour samples (N = 40 patients, 20 with EGFR amplification). Results: In the setting of glioblastoma recurrence, the EGFR pathway was overexpressed regardless of EGFR amplification status, suggesting a common genomic endpoint in recurrent glioblastoma, although EGFR amplification did associate with higher EGFR mRNA expression. Three of forty patients in the study cohort had EGFR-amplified tumours and received targeted EGFR therapy. Their molecular subtypes and clinical outcomes did not significantly differ from patients who received conventional chemotherapy. Conclusion: Our findings suggest that while the EGFR amplification may confer a unique molecular profile in primary glioblastoma, pathway analysis reveals upregulation of the EGFR pathway in recurrence, regardless of amplification status. As such, the EGFR pathway may be a key mediator of glioblastoma progression.
Project description:COLO 320 (Human colorectal cancer cell line) xenograft: Control (Xenograft without rat mesenchymal stem cells (MSCs)) vs Xenograft with rat MSCs