scholarly journals Establishment and optimization of genome-wide CRISPR/Cas9-sgRNA screening system in THP1 cell line for functional oncogenes and tumor suppressor genes

2016 ◽  
Vol 46 (7) ◽  
pp. 839-850
Author(s):  
Tao CHENG ◽  
XiaoBing ZHANG ◽  
Chen CHEN ◽  
Sha HAO ◽  
JianPing ZHANG ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Line ◽  
Tumor Suppressor Genes ◽  
Screening System ◽  
Thp1 Cell ◽  
Suppressor Genes ◽  
Genome Wide

scholarly journals Transposon Mutagenesis-Guided CRISPR/Cas9 Screening Strongly Implicates Dysregulation of Hippo/YAP Signaling in Malignant Peripheral Nerve Sheath Tumor Development

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1584
Author(s):  
Germán L. Vélez-Reyes ◽  
Nicholas Koes ◽  
Ji Hae Ryu ◽  
Gabriel Kaufmann ◽  
Mariah Berner ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Schwann Cell ◽  
Peripheral Nerve ◽  
Cell Line ◽  
Schwann Cells ◽  
Tumor Suppressor Genes ◽  
Tumor Formation ◽  
Loss Of Function ◽  
Suppressor Genes ◽  
Nerve Sheath

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive, genomically complex, have soft tissue sarcomas, and are derived from the Schwann cell lineage. Patients with neurofibromatosis type 1 syndrome (NF1), an autosomal dominant tumor predisposition syndrome, are at a high risk for MPNSTs, which usually develop from pre-existing benign Schwann cell tumors called plexiform neurofibromas. NF1 is characterized by loss-of-function mutations in the NF1 gene, which encode neurofibromin, a Ras GTPase activating protein (GAP) and negative regulator of RasGTP-dependent signaling. In addition to bi-allelic loss of NF1, other known tumor suppressor genes include TP53, CDKN2A, SUZ12, and EED, all of which are often inactivated in the process of MPNST growth. A sleeping beauty (SB) transposon-based genetic screen for high-grade Schwann cell tumors in mice, and comparative genomics, implicated Wnt/β-catenin, PI3K-AKT-mTOR, and other pathways in MPNST development and progression. We endeavored to more systematically test genes and pathways implicated by our SB screen in mice, i.e., in a human immortalized Schwann cell-based model and a human MPNST cell line, using CRISPR/Cas9 technology. We individually induced loss-of-function mutations in 103 tumor suppressor genes (TSG) and oncogene candidates. We assessed anchorage-independent growth, transwell migration, and for a subset of genes, tumor formation in vivo. When tested in a loss-of-function fashion, about 60% of all TSG candidates resulted in the transformation of immortalized human Schwann cells, whereas 30% of oncogene candidates resulted in growth arrest in a MPNST cell line. Individual loss-of-function mutations in the TAOK1, GDI2, NF1, and APC genes resulted in transformation of immortalized human Schwann cells and tumor formation in a xenograft model. Moreover, the loss of all four of these genes resulted in activation of Hippo/Yes Activated Protein (YAP) signaling. By combining SB transposon mutagenesis and CRISPR/Cas9 screening, we established a useful pipeline for the validation of MPNST pathways and genes. Our results suggest that the functional genetic landscape of human MPNST is complex and implicate the Hippo/YAP pathway in the transformation of neurofibromas. It is thus imperative to functionally validate individual cancer genes and pathways using human cell-based models, to determinate their role in different stages of MPNST development, growth, and/or metastasis.

scholarly journals Thymoquinone-Induced Reactivation of Tumor Suppressor Genes in Cancer Cells Involves Epigenetic Mechanisms

2019 ◽  
Vol 12 ◽  
pp. 251686571983901 ◽  
Author(s):  
Shahad A Qadi ◽  
Mohammed A Hassan ◽  
Ryan A Sheikh ◽  
Othman AS Baothman ◽  
Mazin A Zamzami ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Suppressor ◽  
Cancer Cells ◽  
Cell Line ◽  
Rna Sequencing ◽  
Cancer Cell ◽  
Tumor Suppressor Genes ◽  
Jurkat Cells ◽  
Leukemia Cell Line ◽  
Suppressor Genes

The epigenetic silencing of tumor suppressor genes (TSGs) is a common finding in several solid and hematological tumors involving various epigenetic readers and writers leading to enhanced cell proliferation and defective apoptosis. Thymoquinone (TQ), the major biologically active compound of black seed oil, has demonstrated anticancer activities in various tumors by targeting several pathways. However, its effects on the epigenetic code of cancer cells are largely unknown. In the present study, we performed RNA sequencing to investigate the anticancer mechanisms of TQ-treated T-cell acute lymphoblastic leukemia cell line (Jurkat cells) and examined gene expression using different tools. We found that many key epigenetic players, including ubiquitin-like containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1 ( UHRF1), DNMT1,3A,3B, G9A, HDAC1,4,9, KDM1B, and KMT2A,B,C,D,E, were downregulated in TQ-treated Jurkat cells. Interestingly, several TSGs, such as DLC1, PPARG, ST7, FOXO6, TET2, CYP1B1, SALL4, and DDIT3, known to be epigenetically silenced in various tumors, including acute leukemia, were upregulated, along with the upregulation of several downstream pro-apoptotic genes, such as RASL11B, RASD1, GNG3, BAD, and BIK. Data obtained from RNA sequencing were confirmed using quantitative reverse transcription polymerase chain reaction (RT-qPCR) in Jurkat cells, as well as in a human breast cancer cell line (MDA-MB-468 cells). We found that the decrease in cell proliferation and in the expression of UHRF1, DNMT1, G9a, and HDAC1 genes in both cancer cell (Jurkat cells and MDA-MB-468 cells) lines depends on the TQ dose. Our results indicate that the use of TQ as an epigenetic drug represents a promising strategy for epigenetic therapy for both solid and blood tumors by targeting both DNA methylation and histone post-translational modifications.

scholarly journals Differential genome-wide array–based methylation profiles in prognostic subsets of chronic lymphocytic leukemia

Blood ◽  
2010 ◽  
Vol 115 (2) ◽  
pp. 296-305 ◽  
Author(s):  
Meena Kanduri ◽  
Nicola Cahill ◽  
Hanna Göransson ◽  
Camilla Enström ◽  
Fergus Ryan ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Lymphocytic Leukemia ◽  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Lymphocytic Leukemia ◽  
Chain Reaction ◽  
Global Methylation ◽  
Suppressor Genes ◽  
Genome Wide ◽  
Polymerase Chain

Abstract Global hypomethylation and regional hypermethylation are well-known epigenetic features of cancer; however, in chronic lymphocytic leukemia (CLL), studies on genome-wide epigenetic modifications are limited. Here, we analyzed the global methylation profiles in CLL, by applying high-resolution methylation microarrays (27 578 CpG sites) to 23 CLL samples, belonging to the immunoglobulin heavy-chain variable (IGHV) mutated (favorable) and IGHV unmutated/IGHV3-21 (poor-prognostic) subsets. Overall, results demonstrated significant differences in methylation patterns between these subgroups. Specifically, in IGHV unmutated CLL, we identified methylation of 7 known or candidate tumor suppressor genes (eg, VHL, ABI3, and IGSF4) as well as 8 unmethylated genes involved in cell proliferation and tumor progression (eg, ADORA3 and PRF1 enhancing the nuclear factor-κB and mitogen-activated protein kinase pathways, respectively). In contrast, these latter genes were silenced by methylation in IGHV mutated patients. The array data were validated for selected genes using methylation-specific polymerase chain reaction, quantitative reverse transcriptase–polymerase chain reaction, and bisulfite sequencing. Finally, the significance of DNA methylation in regulating gene promoters was shown by reinducing 4 methylated tumor suppressor genes (eg, VHL and ABI3) in IGHV unmutated samples using the methyl-inhibitor 5-aza-2′-deoxycytidine. Taken together, our data for the first time reveal differences in global methylation profiles between prognostic subsets of CLL, which may unfold epigenetic silencing mechanisms involved in CLL pathogenesis.

scholarly journals Decitabine-Induced Changes in Human Myelodysplastic Syndrome Cell Line SKM-1 Are Mediated by FOXO3A Activation

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Wen Zeng ◽  
Hanjun Dai ◽  
Ming Yan ◽  
Xiaojun Cai ◽  
Hong Luo ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Line ◽  
Tumor Suppressor Genes ◽  
Therapeutic Effects ◽  
Suppressor Genes ◽  
Recommended Treatment ◽  
Cycle Arrest ◽  
Induced Changes ◽  
Hyperphosphorylated Form

The epigenetic silencing of tumor suppressor genes in myelodysplastic syndromes (MDS) can potentially confer a growth advantage to individual cellular clones. Currently, the recommended treatment for patients with high-risk MDS is the methylation agent decitabine (DAC), a drug that can induce the reexpression of silenced tumor suppressor genes. We investigated the effects of DAC treatment on the myeloid MDS cell line SKM-1 and investigated the role of FOXO3A, a potentially tumor-suppressive transcription factor, by silencing its expression prior to DAC treatment. We found that FOXO3A exists in an inactive, hyperphosphorylated form in SKM-1 cells, but that DAC both induces FOXO3A expression and reactivates the protein by reducing its phosphorylation level. Furthermore, we show that this FOXO3A activation is responsible for the DAC-induced differentiation of SKM-1 cells into monocytes, as well as for SKM-1 cell cycle arrest, apoptosis, and autophagy. Collectively, these results suggest that FOXO3A reactivation may contribute to the therapeutic effects of DAC in MDS.

Abstract 840: Identification of novel colorectal tumor suppressor genes through genome-wide promoter hypermethylation analysis

2019 ◽  
Author(s):  
Sarah Bazzocco ◽  
Jose Higinio Dopeso ◽  
Águeda Martínez-Barriocanal ◽  
Estefanía Anguita ◽  
Rocio Nieto ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Promoter Hypermethylation ◽  
Colorectal Tumor ◽  
Suppressor Genes ◽  
Genome Wide

scholarly journals Genome-Wide Methylation Analysis and Epigenetic Unmasking Identify Tumor Suppressor Genes in Hepatocellular Carcinoma

2013 ◽  
Vol 145 (6) ◽  
pp. 1424-1435.e25 ◽  
Author(s):  
Kate Revill ◽  
Tim Wang ◽  
Anja Lachenmayer ◽  
Kensuke Kojima ◽  
Andrew Harrington ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Methylation Analysis ◽  
Suppressor Genes ◽  
Genome Wide

scholarly journals Genome-Wide DNA Methylation Indicates Silencing of Tumor Suppressor Genes in Uterine Leiomyoma

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e33284 ◽  
Author(s):  
Antonia Navarro ◽  
Ping Yin ◽  
Diana Monsivais ◽  
Simon M. Lin ◽  
Pan Du ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Uterine Leiomyoma ◽  
Suppressor Genes ◽  
Genome Wide
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