Storr, Sarah
(2022)
Analysis of transcriptomic changes that occur following loss of DARPP-32 expression in breast cancer.
MRes thesis, University of Nottingham.
Abstract
Background
Breast cancer is the most common cancer in the UK, with >55,000 new cases per annum and >11,500 deaths, with metastasis the major cause of mortality. Since the early 1990s, the incidence of breast cancer has increased by 18% in the UK with cases predicted to rise 2% between 2014 and 2035. The molecular basis that underpins cancer progression and metastasis is complex and presents an important research challenge. Dopamine and cAMP-regulated protein phosphatase (DARPP-32) is a central signalling switch in the brain and the consequence of altered phosphorylation has been extensively studied. In breast cancer, we have demonstrated that low expression of DARPP-32 is associated with poor clinical outcome of patients; however, the mechanism by which DARPP-32 exerts this effect remains unclear.
Methods
DARPP-32 expression was reduced in oestrogen receptor positive luminal B breast cancer cells (T47D) using siRNA, and both DARPP-32 knockdown T47D cells, negative control siRNA treated control T47D cells were subject to stimulation with estradiol17β (E2) or inhibition of protein kinase A (PKA) with PKA inhibitor fragment (6-22) amide prior to RNA sequencing. A bioinformatics pipeline of Trim Galore, Kallisto, DESeq2 was utilised and pathway overrepresentation analysis was performed using Qiagen Ingenuity Pathway Analysis software.
Results
A reduction in DARPP-32 expression resulted in the differential expression of 202 transcripts, with the top 10 downregulated genes being CLN8, GRAMD2B, HNRNPC, ZCCHC7, CMIP, OSBPL2, TSEN34, MAN2C1, NDUFS1, and MAP2 and the top 10 upregulated genes being KBTBD11, ZNF230, BCL2L11, ZNF227, VKORC1L1, C19orf25, TSEN35, SART3, AAGAB and CHEK2. 42641 differentially expressed transcripts were identified following E2 stimulation of DARPP-32 knockdown cells, 40065 differentially expressed transcripts were identified when control cells were stimulated with E2, and 193 differentially expressed transcripts were identified when DARPP-32 knockdown cells were compared with control cells, when both were subject to E2 stimulation. When DARPP-32 knockdown cells and control cells were both treated with E2, the top 10 downregulated genes were BCLAF1, NEDD4L, MAGEA3, RBCK1, PTBP1, GON7, PBX3, TXLNA, and METTL8 and the top 10 upregulated genes were CTDSPL, ARMC1, FGFR3, G3BP1, TNRC6A, EIF3L, CDKN1C, PUF60, BMPR1B, and SLC16A7.
When a PKA inhibitor was used to treat DARPP-32 knockdown cells and/or control cells, 1155 differentially expressed transcripts were identified in DARPP-32 knockdown cells when treated with PKAi, 1704 differentially expressed transcripts were identified when control cells were treated with PKAi, and 181 differentially identified transcripts were identified when DARPP-32 knockdown cells were compared with control cells, when both were subject to PKA inhibition.
Conclusions
The transcriptomic changes resultant from DARPP-32 knockdown in ER positive breast cancer cells have been determined, with and without E2 stimulation and PKA inhibition. These changes identify that DARPP-32 functions as an important partner in many cancer-associated signalling pathways that have been linked with poor prognosis and clinical outcome of breast cancer patients.
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