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CRISPR/Cas9-Based Approaches for Investigating Mechanisms of Oncogene Activation and Tumor Suppression

Drainas, Alexandros Panagiotis

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Abstract

Cancer is largely a disease of the genome. Cancer development is thought to involve the gradual acquisition of mutations that can activate oncogenes and/or inactivate tumor suppressor genes, resulting in a series of genetic changes that stimulate growth, attenuate cell death, destroy checkpoint controls, promote further genetic instability, and enable metastasis.

In the first part of my thesis, I focused on deciphering how activation of oncogenes by structural copy number alterations (SCNAs) that relocate enhancers in close proximity to oncogenes can be achieved, rather than activation by mutation or amplification. This mechanism was recently described as enhancer hijacking (EHJ). I contributed to the understanding of the EHJ mechanism at two genomic loci, IGF2, a known oncogenic locus in colorectal cancer and IRS4, a gene identified as a top pan-cancer EHJ candidate. To achieve this, I recapitulated the rearrangements associated with EHJ in colorectal and lung cell lines using the CRISPR/Cas9 genomic engineering system and tested for IGF2 and IRS4 overexpression, respectively. The rearrangements were successfully reconstructed; however an increase in gene expression was not achieved, suggesting a more complex mechanism of activation or context-dependency than initially anticipated. Investigation of the tumor promoting role of IRS4 was supported using mouse xenografts, where constitutive overexpression of IRS4 leads to formation of larger tumors in comparison to control tumors.

In the second part of this thesis I emphasized on the identification of genes, which, when disrupted, lead to sustained cell growth and can be potential tumor suppressors. To achieve this, I employed systematic screens on cells with different genetic backgrounds using a combination of CRISPR/Cas9-based whole genome knockout libraries and the powerful anchorage independent growth assay. I was able to verify known tumor suppressor genes, which include components of the Hippo and mTOR pathways, as well as to identify novel candidates including FRYL and AHR. Furthermore, a growth screen under non-selecting conditions was performed and identified numerous candidates found in the initial anchorage independent growth screen, which further supports the growth promoting roles of the candidate genes. In conclusion, in my study I identified potential tumor suppressors that lead untransformed cells to enhanced as well as anchorage independent growth.

Item Type: Dissertation
Supervisor: Lyko, Prof. Dr. Frank
Date of thesis defense: 25 November 2016
Date Deposited: 02 Jan 2017 13:55
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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