Investigating ARID1A in prostate cancer

Hartley, Andrew (2022) Investigating ARID1A in prostate cancer. PhD thesis, University of Glasgow.

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Abstract

Prostate cancer (PC) is the most common cancer in men in the developed world, and the second leading cause of cancer death in the U.K. Most of these deaths are caused by the advanced and metastatic forms of this disease which have no curative options. Understanding and targeting metastatic prostate cancer remains one of the primary research goals in prostate cancer research.

The Sleeping Beauty screen used a forward-mutagenesis transposon-based system to try to identify novel drivers of prostate cancer which cooperated with loss of Pten in vivo. This screen showed that reduced Arid1a expression correlated with a poorer survival and elevated metastasis in vivo. ARID1A functions as part of the BAF chromatin remodelling complex, an epigenetic regulator of gene expression which controls chromatin accessibility.

Using a genetically engineered mouse (GEM) model, we were able to delete Arid1a and Pten from the mouse prostate genome to investigate the genetic interaction of these two genes. This showed that Pb-Cre+ve Ptenfl/fl Arid1afl/fl mice developed clinical endpoint tumours very rapidly compared to Pb-Cre+ve Ptenfl/fl Arid1a+/+ (median 4 months vs 10 months respectively). These Pb-Cre+ve Ptenfl/fl Arid1afl/fl tumours also have an invasive phenotype. Pb-Cre+ve Ptenfl/fl Arid1afl/+ tumours however showed no significant change in survival compared to Pb-Cre+ve Ptenfl/fl Arid1a+/+ .

RNA-Seq and ChIP-Seq were performed to understand how loss of Arid1a influenced tumorigenesis in our GEM model. This showed that following loss of Arid1a, tumours upregulated growth and cell cycle signalling pathways. Ki67, a marker of proliferation, confirmed that Pb-Cre+ve Ptenfl/fl Arid1afl/fl tumours had a higher rate of proliferation compared to Pb-Cre+ve Ptenfl/fl Arid1a+/+ tumours. ChIP-Seq findings suggested that Arid1a directly binds genes involve in migration and invasion which were subsequently upregulated following loss of Arid1a. This suggested that the increased proliferation may be due to a re-targeting of the BAF complex rather than direct repression of growth by Arid1a itself. Immunohistochemistry (IHC) and GSEA suggests that P53-dependent senescence signalling is still present in Pb-Cre+ve Ptenfl/fl Arid1afl/fl though has likely been bypassed.

Generating ARID1A knockout clones with CRISPR-Cas9 of DU145, a human PC cell line, phenotypically increases their growth and survival following ARID1A loss, though decreases their invasion abilities. RNA-Seq on DU145 ARID1AKO clones shows an elevation in growth and cell cycle signalling following ARID1A loss, though a reduction in migration signalling. RNA-Seq suggests however that these cells may be capable of local extracellular matrix remodelling through matrix metalloprotease upregulation. Comparisons between the GEM model and DU145 show overlapping upregulation in pathways relating to cell cycle and DNAdamage response. DNA damage response findings correlate with clinical data suggest ARID1A mutant PC has high mutational burden.

Data presented in this thesis therefore shows that Arid1a loss in vivo cooperated with Pten loss and produced aggressive and invasive prostate tumours. Loss of Arid1a upregulated growth and cell cycle signalling, though this is likely due to a retargeting of the BAF complex itself as Arid1a was only shown to directly repress genes involved in migration and invasion. Using ARID1AKO PC cell line DU145 also shows an elevation in cell cycle signalling. Understanding how loss of ARID1A causes BAF complex retargeting warrants additional research.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Beatson Institute of Cancer Research
Supervisor's Name: Ahmad, Dr. Imran and Blyth, Professor Karen
Date of Award: 2022
Depositing User: Theses Team
Unique ID: glathesis:2022-83300
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 07 Dec 2022 10:07
Last Modified: 13 Dec 2022 12:14
Thesis DOI: 10.5525/gla.thesis.83300
URI: https://theses.gla.ac.uk/id/eprint/83300

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