Investigating the interaction between PDE4D7 and DHX9 in the progression of prostate cancer

Busiau, Tara Simone (2021) Investigating the interaction between PDE4D7 and DHX9 in the progression of prostate cancer. PhD thesis, University of Glasgow.

Abstract

Prostate cancer (PC) is the most commonly diagnosed disease in men, and is considered the second most likely cause of cancer-related death in the Western male population behind lung cancer. In recent years, the rate of disease detection has significantly increased in part due to the successful implementation of the prostate specific antigen (PSA) testing in clinics. Currently, PC is positively diagnosed when PSA levels are detected to be over 4.0 ng/mL in the blood plasma. However, increasing evidence has shown that this number is often misleading and inaccurate. The prostate is known to have a very good blood supply, therefore small amounts of PSA can always be detected. Furthermore, the PSA reflects the state of the whole gland itself rather than indicating the presence of a tumour at this site. As the availability of PSA testing increases, this has also resulted in the detection of false positives as some men have naturally higher levels of PSA than others. On the other hand, some cancers can go undetected as it may have progressed so far that it no longer expresses PSA. Following a positive diagnosis, the most common course of treatment prescribed is androgen deprivation therapy (ADT). ADT refers to the treatments that aim to reduce the effects of testosterone and other androgens by surgically or chemically preventing their production. Although ADT is known as the gold standard in PC treatment, approximately 15% of men fail to respond to this form of therapy. Furthermore, after a mean time of 13-19 months, some men become castration resistant and no longer respond to ADT.
There is currently a need to identify novel biomarkers in order to accurately diagnose and stage the disease. Furthermore, new drug targets need to be identified in order to provide the best course of treatment. Previous work by the Baillie laboratory, in collaboration with Philips Diagnostics, recently identified phosphodiesterase 4D7 (PDE4D7) to be a novel prognostic marker for disease. PDE4D7 expression was shown to be decreased in PC cell lines and primary tumours as the disease progressed to the hormone independent state. The 3',5'-cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) are known to play a role in disease progression as they mediate downstream signalling pathways that can promote cell growth and disease progression. PDE enzymes
are the only known enzyme family to hydrolyse cAMP, and loss of PDE4D7 expression, in particular, during PC correlates with disease progression. Interestingly, DHX9 has recently been identified as a novel interacting partner for PDE4D7. Work by Dr Ashleigh Byrne identified DHX9 as a novel interactor by using mass spectrometry, and since then DHX9 has become a subject of increased interest in the area of cancer research. DHX9 is an RNA/DNA helicase that is involved in multiple cellular processes, including transcription and maintaining genome stability. DHX9 expression is known to increase as cancers, such as lung and colorectal, progress towards their metastatic stages. Previous work by other lab groups have demonstrated that DHX9 is linked to multiple signalling pathways that are involved in cancer development, such as the mTOR and p53 pathway. Interestingly, DHX9 maps to the PC susceptibility locus, making it an interesting protein to study in the context of PC disease progression and metastasis.
Work in this thesis provides further evidence that PDE4D7 and DHX9 proteins are novel interactors in PC. By using a series of biochemical techniques, PDE4D7 was shown to interact with DHX9 in androgen sensitive PC cell line. By using peptide array technology, I was able to map where this interaction took place and define docking sites on both protein partners. DHX9 was found to bind within the newly identified FLY multi docking site within the upstream conserved region-1 (UCR1) of PDE4D7. Furthermore, PDE4D7 was found to bind within DHX9’s helicase core domain, suggesting that it may play a role in regulating its activity. I was able to further validate these binding sites by designing cell permeable peptides designed to disrupt protein binding in vitro.
Considering that many PKA substrates are found in complex with PDEs, DHX9 was found to be a PKA substrate in vitro. By using biochemical techniques, such as immunoprecipitations and proximity ligations assay, DHX9 was found to be readily phosphorylated by PKA, and this was significantly increased when cells were pre-treated with the adenylate cyclase activator forskolin and the general PDE inhibitor IBMX. By using peptide array technology and bioinformatic predictions, serine 449 within the helicase core of DHX9 was found to be in a PKA motif and phosphorylated by PKA. Interestingly, this serine can be found upstream of the PDE4D7 binding site. Disruption of the interaction between
PDE4D7 and DHX9 led to a significant increase in DHX9 phosphorylation, supporting the idea that PDE4D7 binding regulated DHX9 phosphorylation. Using the information gained in the peptide arrays, a phospho-specific antibody against DHX9 was raised. The newly synthesised antibody was able to detect phosphorylated DHX9 by western blotting and immunofluorescence with confocal microscopy.
Although DHX9 is known to be differentially expressed in multiple cancers, little is known about its function in PC. Silencing of DHX9 expression using siRNA technology significantly inhibited PC cell growth. Interestingly, previous work by Erzikhan et al (2009) demonstrated that inhibiting DHX9’s oncogenic activity, by disrupting DHX9’s interaction with EWS-FL1 using YK-4-279, significantly decreased growth when assessed by xCELLigence technology. Unfortunately, disruption of this interaction between PDE4D7 and DHX9 did not alter the growth of PC cells following treatment with the cell permeable disruptor peptide. However, the disruption of the interaction between PDE4D7 and DHX9 significantly decreased DHX9’s ability to promote R-loop formation when assessed by immunofluorescence and confocal microscopy. Using RPPA analysis, I was also able to show that the loss of DHX9 expression in PC cells can potentially affect the mTOR signalling pathway.
To conclude, this thesis provides further evidence that PDE4D7 and DHX9 form a signalling complex that may be relevant in PC. I was able to map where these interactions took place and design cell permeable peptides that were able to disrupt this interaction. I was also able to show that DHX9 can be readily phosphorylated in vitro, and its phosphorylation is partly regulated by its interaction with PDE4D7. Loss of DHX9 expression, and interaction with EWS-FL1, significantly decreases cell growth, and this may partly be due to changes in mTOR pathways. However, future work on this topic is still needed. Unfortunately, due to time constraints, the functional implications of DHX9 phosphorylation was not studied in this thesis and I was not able to use relevant human samples to further validate the relevance of my findings.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: PDE4D7, DHX9, prostate cancer, cell signalling.
• Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Supervisor's Name: Baillie, Prof. George S.
• Date of Award: 2021
• Depositing User: Dr Tara S Busiau
• Unique ID: glathesis:2021-81899
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 08 Jan 2021 08:37
• Last Modified: 12 Jan 2021 15:06
• Thesis DOI: 10.5525/gla.thesis.81899
• URI: https://theses.gla.ac.uk/id/eprint/81899

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