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dc.contributor.authorKounatidou, Evangelia Eirini-
dc.descriptionPhD Thesisen_US
dc.description.abstractProstate cancer resistance to AR targeted therapies due to the emergence of AR point mutations and AR splice variants that cannot be targeted by the currently available agents comprise a major clinical challenge. There is paucity of models that accurately reflect the mechanisms of AR regulation in advanced disease. This highlights the high demand of generating novel disease relevant models. A CRISPR pipeline was developed to generate cell line models which harbour specific point mutations in the LBD of AR as well as stop codons in AR exon 5 which resulted in AR-FL knock-out so that the remaining endogenous AR-Vs could be studied discriminately of interfering AR-FL. Using a streptavidin-tagged Cas9 in conjugation with a biotinylated donor template resulted in high donor template knock-in efficiencies and yielded (i) an ARW741L CWR22Rv1 cell line derivative and (ii) an AR-FL knock-out cell line derivative called CWR22Rv1-AR-EK (Exon Knock-out). CWR22Rv1-AR-EK cells retained all endogenous AR-Vs following AR gene editing. AR-Vs acted unhindered following AR-FL deletion to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrated that AR-Vs drive expression of a cohort of cell cycle and DNA damage response genes and depletion of AR-Vs sensitised cells to ionising radiation. To date, elimination of AR-Vs by pharmacological inhibition remains challenging. However, disruption of AR pre-mRNA splicing is nowadays a highly attractive option. A CRISPR-based approach, called CRIME (Cas9-directed Rapid Immunoprecipitation Mass Spectrometry of Endogenous proteins) was developed to isolate and identify the AR-V7-specific spliceosome, in association with a nuclease deficient Cas9 at AR cryptic exon 3. Mass spec-derived hits were screened for their ability to alter AR-V7 mRNA levels in CWR22Rv1 cells. SRSF3 was identified as a potential AR-V7 splicer which promotes cryptic exon 3 skipping in normal prostate. In CRPC, SRSF3 is significantly downregulated and hence cryptic exon 3 inclusion in the mature AR-V7 mRNA transcript is permitted leading to generation of AR-V7.en_US
dc.publisherNewcastle Universityen_US
dc.titleGeneration of CRISPR engineered prostate cancer cell line models to study androgen receptor signalling in advanced prostate canceren_US
Appears in Collections:Northern Institute for Cancer Research

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