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

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.

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Modification of Epigenetic Histone Acetylation in Hepatocellular Carcinoma

Cancers ◽

10.3390/cancers10010008 ◽

2018 ◽

Vol 10 (1) ◽

pp. 8 ◽

Cited By ~ 22

Author(s):

Kwei-Yan Liu ◽

Li-Ting Wang ◽

Shih-Hsien Hsu

Keyword(s):

Hepatocellular Carcinoma ◽

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Chemical Sensor ◽

Enzyme Level ◽

Tumor Therapy ◽

Tumor Formation ◽

Current Evidence ◽

Epigenetic Changes ◽

Suppressor Genes

Cells respond to various environmental factors such as nutrients, food intake, and drugs or toxins by undergoing dynamic epigenetic changes. An imbalance in dynamic epigenetic changes is one of the major causes of disease, oncogenic activities, and immunosuppressive effects. The aryl hydrocarbon receptor (AHR) is a unique cellular chemical sensor present in most organs, and its dysregulation has been demonstrated in multiple stages of tumor progression in humans and experimental models; however, the effects of the pathogenic mechanisms of AHR on epigenetic regulation remain unclear. Apart from proto-oncogene activation, epigenetic repressions of tumor suppressor genes are involved in tumor initiation, procession, and metastasis. Reverse epigenetic repression of the tumor suppressor genes by epigenetic enzyme activity inhibition and epigenetic enzyme level manipulation is a potential path for tumor therapy. Current evidence and our recent work on deacetylation of histones on tumor-suppressive genes suggest that histone deacetylase (HDAC) is involved in tumor formation and progression, and treating hepatocellular carcinoma with HDAC inhibitors can, at least partially, repress tumor proliferation and transformation by recusing the expression of tumor-suppressive genes such as TP53 and RB1.

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Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.1387-1395.1992 ◽

1992 ◽

Vol 12 (3) ◽

pp. 1387-1395

Author(s):

M C Goyette ◽

K Cho ◽

C L Fasching ◽

D B Levy ◽

K W Kinzler ◽

...

Keyword(s):

Colorectal Cancer ◽

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Loss Of Function ◽

Chromosome 18 ◽

Chromosome 5 ◽

Suppressor Genes ◽

Single Defect

Carcinogenesis is a multistage process that has been characterized both by the activation of cellular oncogenes and by the loss of function of tumor suppressor genes. Colorectal cancer has been associated with the activation of ras oncogenes and with the deletion of multiple chromosomal regions including chromosomes 5q, 17p, and 18q. Such chromosome loss is often suggestive of the deletion or loss of function of tumor suppressor genes. The candidate tumor suppressor genes from these regions are, respectively, MCC and/or APC, p53, and DCC. In order to further our understanding of the molecular and genetic mechanisms involved in tumor progression and, thereby, of normal cell growth, it is important to determine whether defects in one or more of these loci contribute functionally in the progression to malignancy in colorectal cancer and whether correction of any of these defects restores normal growth control in vitro and in vivo. To address this question, we have utilized the technique of microcell-mediated chromosome transfer to introduce normal human chromosomes 5, 17, and 18 individually into recipient colorectal cancer cells. Additionally, chromosome 15 was introduced into SW480 cells as an irrelevant control chromosome. While the introduction of chromosome 17 into the tumorigenic colorectal cell line SW480 yielded no viable clones, cell lines were established after the introduction of chromosomes 15, 5, and 18. Hybrids containing chromosome 18 are morphologically similar to the parental line, whereas those containing chromosome 5 are morphologically distinct from the parental cell line, being small, polygonal, and tightly packed. SW480-chromosome 5 hybrids are strongly suppressed for tumorigenicity, while SW480-chromosome 18 hybrids produce slowly growing tumors in some of the animals injected. Hybrids containing the introduced chromosome 18 but was significantly reduced in several of the tumor reconstitute cell lines. Introduction of chromosome 5 had little to no effect on responsiveness, whereas transfer ot chromosome 18 restored responsiveness to some degree. Our findings indicate that while multiple defects in tumor suppressor genes seem to be required for progression to the malignant state in colorectal cancer, correction of only a single defect can have significant effects in vivo and/or in vitro.

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

Epigenetics Insights ◽

10.1177/2516865719839011 ◽

2019 ◽

Vol 12 ◽

pp. 251686571983901 ◽

Cited By ~ 9

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.

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

Journal of Immunology Research ◽

10.1155/2017/4302320 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 5

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.

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Spatially- and temporally-controlled postnatal p53 knockdown cooperates with embryonic Schwann cell precursor Nf1 gene loss to promote malignant peripheral nerve sheath tumor formation

Oncotarget ◽

10.18632/oncotarget.7232 ◽

2016 ◽

Vol 7 (7) ◽

pp. 7403-7414 ◽

Cited By ~ 13

Author(s):

Angela C. Hirbe ◽

Sonika Dahiya ◽

Dinorah Friedmann-Morvinski ◽

Inder M. Verma ◽

D. Wade Clapp ◽

...

Keyword(s):

Schwann Cell ◽

Peripheral Nerve ◽

Gene Loss ◽

Tumor Formation ◽

Nerve Sheath Tumor ◽

Cell Precursor ◽

Peripheral Nerve Sheath Tumor ◽

Nerve Sheath ◽

Nf1 Gene

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ALV-J and REV synergistically activate a new oncogene of KIAA1199 via NF-κB and EGFR signaling regulated by miR-147

10.1101/338244 ◽

2018 ◽

Author(s):

Defang Zhou ◽

Jingwen Xue ◽

Pingping Zhuang ◽

Xiyao Cui ◽

Shuhai He ◽

...

Keyword(s):

Tumor Suppressor ◽

Molecular Mechanism ◽

Tumor Suppressor Genes ◽

Large Scale ◽

Molecular Mechanisms ◽

Tumor Formation ◽

Signalling Pathway ◽

Suppressor Genes ◽

Pathway Crosstalk ◽

Pathogenic Effects

AbstractThe tumorigenesis is the result of the accumulation of multiple oncogenes and tumor suppressor genes changes. Co-infection of avian leucosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV), as two oncogenic retroviruses, showed synergistic pathogenic effects characterized by enhanced tumor initiation and progression. The molecular mechanism underlying synergistic effects of ALV-J and REV on the neoplasia remains unclear. Here, we found co-infection of ALV-J and REV enhanced the ability of virus infection, increased viral life cycle, maintained cell survival and enhanced tumor formation. We combined the high-throughput proteomic readout with a large-scale miRNA screening to identify which molecules are involved in the synergism. Our results revealed co-infection of ALV-J and REV activated a latent oncogene of KIAA1199 and inhibited the expression of tumor suppressor miR-147. Further, enhanced KIAA1199, down-regulated miR-147, activated NF-κB and EGFR were demonstrated in co-infected tissues and tumor. Mechanistically, we showed ALV-J and REV synergistically enhanced KIAA1199 by activation of NF-κB and EGFR signalling pathway, and the suppression of tumor suppressor miR-147 was contributed to maintain the NF-κB/KIAA1199/EGFR pathway crosstalk by targeting the 3’UTR region sequences of NF-κB p50 and KIAA1199. Our results contributed to the understanding of the molecular mechanisms of viral synergistic tumorgenesis, which provided the evidence that suggested the synergistic actions of two retroviruses could result in activation of latent pro-oncogenes.Author summaryThe tumorigenesis is the result of the accumulation of multiple oncogenes and tumor suppressor genes changes. Co-infection with ALV-J and REV showed synergistic pathogenic effects characterized by enhanced tumor progression, however, the molecular mechanism on the neoplasia remains unclear. Our results revealed co-infection of ALV-J and REV promotes tumorigenesis by both induction of a latent oncogene of KIAA1199 and suppression of the expression of tumor suppressor miR-147. Mechanistic studies revealed that ALV-J and REV synergistically enhance KIAA1199 by activation of NF-κB and EGFR signalling pathway, and the suppression of tumor suppressor miR-147 was contributed to maintain the NF-κB/KIAA1199/EGFR pathway crosstalk by targeting the 3’UTR region sequences of NF-κB p50 and KIAA1199. These results provided the evidence that suggested the synergistic actions of two retroviruses could result in activation of latent pro-oncogenes, indicating the potential preventive target and predictive factor for ALV-J and REV induced tumorigenesis.

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Prostaglandin E2 metabolism is activated in Schwann cell lines derived from human NF1 malignant peripheral nerve sheath tumors

Neuron Glia Biology ◽

10.1017/s1740925x04000262 ◽

2004 ◽

Vol 1 (2) ◽

pp. 149-155 ◽

Cited By ~ 5

Author(s):

GAIL D. DEADWYLER ◽

IAN DANG ◽

JULIE NELSON ◽

MAYA SRIKANTH ◽

GEORGE H. DE VRIES

Keyword(s):

Schwann Cell ◽

Peripheral Nerve ◽

Schwann Cells ◽

Cell Lines ◽

Genetic Disorder ◽

Prostaglandin E ◽

Nerve Sheath Tumors ◽

Peripheral Nerve Sheath Tumors ◽

Nerve Sheath ◽

Cox 2

Malignant peripheral nerve sheath tumors (MPNSTs) are characteristic of Neurofibromatosis type 1 (NF1), a human genetic disorder affecting approximately 1 in 3000 individuals. The absence of neurofibromin in Schwann cells results in hyperactivation of Ras, which contributes to Schwann cell hyperplasia. However, additional intracellular abnormalities in Schwann cells might contribute to the malignancy. We now report that cell lines derived from MPNSTs secrete elevated levels of prostaglandin E2 (PGE2), express higher levels of phosphorylated mitogen-activated protein kinase (MAPK), phosphorylated cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2) when compared to normal adult human Schwann cells (nhSCs). PCR analysis reveals that NF1 MPNST cell lines express mRNA for both EP2 and EP4 prostaglandin E2 receptors, whereas nhSCs express only the EP4 receptor. COX-2 inhibitors and PGE2 receptor antagonists decrease the proliferation of MPNST cell lines. These results indicate that prostaglandin metabolism is activated in MPNSTs and might contribute to tumor growth in NF1.

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