In vitro co-targeting of PARP-1 and HDAC-1 in prostate cells of varying maspin status

Toomey, Jasmine A. (2020) In vitro co-targeting of PARP-1 and HDAC-1 in prostate cells of varying maspin status. Doctoral thesis, Northumbria University.

[img]
Preview
Text (Doctoral Thesis)
toomey.jasmine_phd.pdf - Submitted Version

Download (31MB) | Preview

Abstract

Prostate cancer is the most common form of cancer in males. In the UK, 1 in 8 males will develop prostate cancer in their lifetime, making research into this condition important. Current research is focused on the identification and development of new therapeutic targets and the repurposing of existing drugs for cancer therapy; to specifically exploit aggressive tumours associated with poor prognosis.
Constitutive activation of the inflammatory transcription factor Nuclear factor kappa-light-chainenhancer of activated B cells (NF-kB) and reduced expression of the tumour suppressor maspin have both been implicated in prostate cancer progression. Poly (ADP-ribose) polymerase-1 (PARP-1), a DNA strand break repair protein, is a known transcriptional co-regulator of NF-kB. Continuous activation of PARP-1 is implicated in increased inflammatory signalling, cellular dysfunction and consequently tumour progression. Previous studies have shown that inhibition of NF-kB using PARP inhibitors can sensitise tumour cells to chemotherapeutic agents. Histone deacetylases (HDAC) play a role in the modification and regulation of gene expression and HDAC-1 is highly expressed and activated in metastatic prostate cancer. It is also involved in the repair of DNA double strand breaks by Homologous recombination (HR). Maspin is an endogenous inhibitor of HDAC-1 and studies have shown that HDAC-1 inhibitors limit activation of NF-kB, stimulate re-expression of maspin and induce a “BRCAness phenotype”. To this end, combined inhibition of PARP-1 and HDAC-1 to target NF-kB and HDAC-1 may serve as a novel therapy in tumours which are deficient in maspin.
The interactive effects between NF-kB, PARP-1 and maspin on the cellular behaviours of prostate cancer cells proficient (PC3) or depleted (DU145) for maspin were investigated in the presence or absence of specific inhibitors of PARP-1 (Rucaparib) and HDAC-1 (Trichostatin A), alone and in combination. A normal prostate cell line (PNT1A) proficient for maspin was also used. Mouse embryonic fibroblasts (MEF) proficient and deficient for NF-kB subunits were utilised to compare maspin expression levels and investigate sensitivity to treatments. siRNA was also used in mechanistic studies to explore the roles of maspin and PARP-1 in cell survival following treatment with Rucaparib and TSA, alone and in combination.
Rucaparib as a single agent significantly reduced survival in DU145 prostate cancer cells depleted in maspin, compared to PNT1A and PC3 prostate cells proficient for maspin. DU145 cells expressed reduced levels of PARP-1 protein and increased levels of IKKα. MEF cells deficient for IKKα expressed maspin and were less sensitive to Rucaparib than MEF cells expressing IKKα. Maspin silencing by siRNA in cells proficient for maspin reduced PARP-1 expression levels and enhanced cell sensitivity to Rucaparib. PARP-1 silencing by siRNA in DU145 and PC3 cells expressing differential levels of maspin did not significantly affect sensitivity to Rucaparib. These data indicate that increased sensitivity to Rucaparib is likely independent to reduced PARP-1 protein levels but may be in part due to increased levels of IKKα and consequently maspin. IKKα may therefore serve as a potential target for cancer therapy and maspin expression may represent a novel predictive biomarker for PARP inhibitor sensitivity in prostate cancers. Combination of TSA with Rucaparib reduced prostate cancer survival, supporting tumour specific sensitivity to co treatment. At GI50 concentrations there was no significant difference in cell sensitivity, following co treatment, between DU145 and PC3 cells, suggesting that sensitivity to co-treatment is independent of IKKα and maspin expression status. Despite a lack of variance in cell survival between DU145 and PC3 cells in the presence of co-treatments at GI50, there was a significant increase in DNA single and double strand break in DU145 cells, compared to PNT1A and PC3 cells. RAD51 expression was reduced in both DU145 and PC3 cells exposed to cotreatment, indicating dysfunctional DNA DSB repair by HR in both of these cell lines. The increased DNA damage response to co-treatment at GI50 in DU145 cells could therefore be attributable to a known BRCA mutation or increased “PARP trapping” at DNA breaks. HDAC inhibitors are known to induce a “BRCAness” phenotype and sensitise cells to DNA repair inhibitors by reducing the capacity to repair DSBs via HR. Loss of HR capacity requires a reliance on the NHEJ pathway for DNA repair and this pathway is upregulated by PARP. Acetylation of PARP-1 in the presence of TSA may lead to the promotion of DNA repair by NHEJ. However, in the presence of Rucaparib, NHEJ is blocked via “PARP trapping” at the site of DNA breaks, resulting in synthetic lethality. Combination of PARP and HDAC inhibitors represent a promising therapeutic strategy for prostate cancer irrespective of BRCA status.

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: prostate cancer, drug repurposing, Rucaparib, Trichostatin A, molecular biology
Subjects: C700 Molecular Biology, Biophysics and Biochemistry
Department: Faculties > Health and Life Sciences > Applied Sciences
University Services > Graduate School > Doctor of Philosophy
Depositing User: John Coen
Date Deposited: 25 Nov 2020 08:45
Last Modified: 25 Nov 2020 08:45
URI: http://nrl.northumbria.ac.uk/id/eprint/44836

Actions (login required)

View Item View Item

Downloads

Downloads per month over past year

View more statistics