Saidy, Behnaz
(2022)
The role of DARPP-32 in breast cancer progression.
PhD thesis, University of Nottingham.
Abstract
Breast cancer accounts for one in every eight females with cancer, and it is the fifth most common cause of cancer death. Previously, our group demonstrated that low expression of DARPP-32 is associated with adverse breast cancer specific survival. The principle aim of this study is to explore the role of DARPP-32 in breast cancer progression.
In this study, in addition to the assessment of mRNA expression within the METABRIC breast cancer cohort, Immunohistochemistry was used in order to assess the expression levels of PKA, PP1, Cdk5, Sp7, GRB7, DKK1 and the phosphorylation of DARPP-32 at Threonine-34 in a large cohort of early-stage primary breast cancer patients.
To investigate DARPP-32 signalling in breast cancer, purvalanol B, tautomycetin, PKA inhibitor fragment (6-22) amide and forskolin were utilised. The effect of compounds on the activity of Cdk5, PP1 and PKA was determined using ELISA, as was the effect on DARPP-32 Threonine-34 and Threonine-75.
RNA sequencing was utilised to analyse transcriptomic changes caused by DARPP-32 knockdown in combination with estradiol17β (E2) stimulation or treatment with PKA inhibitor fragment (6-22) amide.
The effects of combined expression of PKA, PP1, DARPP‐32 and Cdk5 on breast cancer specific survival were assessed. Although PP1 protein expression alone was not associated with patient survival, combined expression of cytoplasmic and nuclear low PP1 and low DARPP‐32, and low PP1 and low Cdk5 were significantly associated with adverse breast cancer specific survival. Low expression levels of PKA and Cdk5, as well the combination of low PKA and low Cdk5 expression were significantly associated with adverse breast cancer specific survival. Low expression levels of DKK1, Sp7 and phosphorylated DARPP-32 at Threonine-34 were significantly associated with adverse breast cancer specific survival whereas low protein expression of GRB7 was significantly associated with improved breast cancer specific survival. In addition, low mRNA expression of Cdk5, PRKAR1A, PRKAR2A, PRKACB, PPP1CB, and PPP1CC were significantly associated with adverse breast cancer specific survival.
MDA-MB-231 cells were significantly sensitive to all drugs (purvalanol B, tautomycetin, PKA inhibitor fragment (6-22) amide and forskolin) at 72 hours. In T47D cells, all compounds were cytotoxic at 24 and 48 hours. In contrast, MCF7 were significantly sensitive to all drugs at 24, 48 and 72 hour time points. Significant reductions in PKA activity was observed following treatment with PKA inhibitor fragment (6-22) amide (P=0.05). Significant reductions in Cdk5 activity were observed following treatment with Cdk5 inhibitor, purvalanol B (P=0.05). Significant reductions in DARPP-32 Threonine-34 phosphorylation were observed following treatment with PP1/PP2A inhibitor, Tautomycetin (P=0.05). Significant reductions in DARPP-32 Threonine-75 phosphorylation were observed following treatment with PKA activator, Forskolin (P=0.05).
DARPP-32 knockdown led to identification of 202 differentially expressed transcripts using RNA sequencing when compared against the control cells; ENST00000443236.6 (CPAMD8), ENST00000521309.5 (YWHAZ), and ENST00000651564.1 (CPAMD8) transcripts were identified as the most significantly altered transcripts.
In summary, all biomarkers were considered to be involved in the development of breast cancer and our data highlights the importance of DARPP-32 as a regulator of cancer associated signalling pathways which may hold a significant therapeutic potential in breast cancer.
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