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The transcriptional modulator ZBED6 regulates cell cycle and growth of human colorectal cancer cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. (MOLCAN)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The transcription factor ZBED6 is a repressor of IGF2 whose action impacts development, cell proliferation and growth in placental mammals. In human colorectal cancers, IGF2 overexpression is mutually exclusive with somatic mutations in PI3K signaling components, providing genetic evidence for a role in the PI3K pathway. To understand the role of ZBED6 in tumorigenesis, we engineered and validated somatic cell ZBED6 knock-outs in the human colorectal cancer cell lines RKO and HCT116. Transcriptome analyses revealed enrichment of cell cycle-related processes among differentially expressed genes in both cell lines. Chromatin immunoprecipitation sequencing analyses displayed enrichment of ZBED6 binding at genes upregulated in ZBED6-/- knockout clones. Ten differentially expressed genes were identified as putative direct gene targets and their downregulation by ZBED6 was experimentally validated. Eight of these genes were linked to the Wnt, Hippo, TGF-b, EGFR or PI3K pathways, all involved in colorectal cancer development. Ablation of ZBED6 affected the cell cycle and led to increased growth rate of ZBED6-/- RKO cells. These observations support a role for transcriptional modulation by ZBED6 in cell cycle regulation and growth of colorectal cancers.

National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-235564OAI: oai:DiVA.org:uu-235564DiVA: diva2:761232
Available from: 2014-11-05 Created: 2014-11-05 Last updated: 2015-02-03
In thesis
1. Understanding Cancer Mutations by Genome Editing
Open this publication in new window or tab >>Understanding Cancer Mutations by Genome Editing
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mutational analyses of cancer genomes have identified novel candidate cancer genes with hitherto unknown function in cancer. To enable phenotyping of mutations in such genes, we have developed a scalable technology for gene knock-in and knock-out in human somatic cells based on recombination-mediated construct generation and a computational tool to design gene targeting constructs. Using this technology, we have generated somatic cell knock-outs of the putative cancer genes ZBED6 and DIP2C in human colorectal cancer cells. In ZBED6-/- cells complete loss of functional ZBED6 was validated and loss of ZBED6 induced the expression of IGF2. Whole transcriptome and ChIP-seq analyses revealed relative enrichment of ZBED6 binding sites at upregulated genes as compared to downregulated genes. The functional annotation of differentially expressed genes revealed enrichment of genes related to cell cycle and cell proliferation and the transcriptional modulator ZBED6 affected the cell growth and cell cycle of human colorectal cancer cells. In DIP2C-/-cells, transcriptome sequencing revealed 780 differentially expressed genes as compared to their parental cells including the tumour suppressor gene CDKN2A. The DIP2C regulated genes belonged to several cancer related processes such as angiogenesis, cell structure and motility. The DIP2C-/-cells were enlarged and grew slower than their parental cells. To be able to directly compare the phenotypes of mutant KRAS and BRAF in colorectal cancers, we have introduced a KRASG13D allele in RKO BRAFV600E/-/-/ cells. The expression of the mutant KRAS allele was confirmed and anchorage independent growth was restored in KRASG13D cells. The differentially expressed genes both in BRAF and KRAS mutant cells included ERBB, TGFB and histone modification pathways. Together, the isogenic model systems presented here can provide insights to known and novel cancer pathways and can be used for drug discovery.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 37 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1054
Keyword
Genome editing, rAAV, ZBED6, DIP2C, KRAS, BRAF, colorectal cancer, tumor driver genes, cancer pathways
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-235680 (URN)978-91-554-9106-2 (ISBN)
Public defence
2014-12-19, Rudbeck Salen, Uppsala University, Rudbeck Laboratory SE-751 85, Uppsala, 09:15 (English)
Opponent
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Available from: 2014-11-27 Created: 2014-11-07 Last updated: 2015-09-11

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CiteExportLink to record
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