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Understanding Cancer Mutations by Genome Editing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. (MOLCAN)
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 [en]
Genome editing, rAAV, ZBED6, DIP2C, KRAS, BRAF, colorectal cancer, tumor driver genes, cancer pathways
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-235680ISBN: 978-91-554-9106-2OAI: oai:DiVA.org:uu-235680DiVA: diva2:761612
Public defence
2014-12-19, Rudbeck Salen, Uppsala University, Rudbeck Laboratory SE-751 85, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-11-27 Created: 2014-11-07 Last updated: 2015-09-11
List of papers
1. Computational and molecular tools for scalable rAAV mediated genome editing
Open this publication in new window or tab >>Computational and molecular tools for scalable rAAV mediated genome editing
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The rapid discovery of potential driver mutations through large scale mutational analyses of human cancers generates a need to characterize their cellular phenotypes. Among the techniques for genome editing, recombinant adeno-associated virus (rAAV) mediated gene targeting is particularly suited to knock-in of single nucleotide substitutions. However, the generation of gene targeting constructs and the targeting process is time consuming and labor-intense. To facilitate rAAV mediated gene targeting, we developed the first software and complementary automation friendly vector tools to generate optimized targeting constructs for editing human protein encoding genes. By computational approaches, rAAV constructs for editing ~72% of bases in protein-coding exons were designed. Similarly, ~81% of genes were predicted to be targetable by rAAV mediated knock-out. A Gateway based cloning system for facile generation of rAAV constructs suitable for robotic automation was developed and used in successful generation of targeting constructs. Together, these tools enable automated rAAV targeting construct design, generation as well as enrichment and expansion of targeted cells with desired integrations.

National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-235563 (URN)
Available from: 2014-11-05 Created: 2014-11-05 Last updated: 2015-02-03
2. The transcriptional modulator ZBED6 regulates cell cycle and growth of human colorectal cancer cells
Open this publication in new window or tab >>The transcriptional modulator ZBED6 regulates cell cycle and growth of human colorectal cancer cells
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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:nbn:se:uu:diva-235564 (URN)
Available from: 2014-11-05 Created: 2014-11-05 Last updated: 2015-02-03
3. DIP2C regulates expression of the tumor suppressor gene CDKN2A
Open this publication in new window or tab >>DIP2C regulates expression of the tumor suppressor gene CDKN2A
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The disco-interacting protein 2 homolog C (DIP2C) gene is an uncharacterized candidate

breast and lung cancer gene. The gene contains a DMAP1 binding domain, pointing to

potential involvement in DNMT1-dependent methylation. To study the role of DIP2C in

tumor development, we engineered human DIP2C knockout cell systems by rAAV-mediated

gene targeting. Homo- and heterozygous RKO DIP2C knockout cells displayed enlarged cells

and growth retardation. This phenotype was most pronounced in DIP2C-/- knockouts, and

these cells also displayed a significant decrease in DIP2C mRNA levels. RNA sequencing

revealed 780 genes affected by the loss of DIP2C, including the cellular senescence marker

P16INK4a. Functional annotation of the regulated genes shows enrichment of genes involved

with cell death processes, cell structure and motility. Furthermore, KEGG pathway analysis

shows association of 19 genes with pathways in cancer. In conclusion, the phenotypic data

and expression changes induced by loss of DIP2C indicate that the gene function may be

important for several biological processes implicated in cancer, and that loss of gene function

may be a trigger of cellular senescence.

National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-235565 (URN)
Available from: 2014-11-05 Created: 2014-11-05 Last updated: 2015-02-03
4. Core Ras Pathway Signaling in Human Colorectal Cancers Revealed by Isogenic Modeling of NF1, KRAS and BRAF Mutations
Open this publication in new window or tab >>Core Ras Pathway Signaling in Human Colorectal Cancers Revealed by Isogenic Modeling of NF1, KRAS and BRAF Mutations
2012 (English)In: European Journal of Cancer, ISSN 0959-8049, E-ISSN 1879-0852, Vol. 48, no Suppl.5, S118-S118 p.Article in journal, Meeting abstract (Refereed) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-194476 (URN)10.1016/S0959-8049(12)71162-0 (DOI)000313036501006 ()
Conference
22nd Biennial Congress of the European-Association-for-Cancer-Research, JUL 07-10, 2012, Barcelona, SPAIN
Available from: 2013-02-15 Created: 2013-02-14 Last updated: 2015-09-11Bibliographically approved

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