Unravelling transcriptomic complexity in breast cancer through modulation of DARPP-32 expression and signalling pathways.

DARPP-32 is a key regulator of protein-phosphatase-1 (PP-1) and protein kinase A (PKA), with its function dependent upon its phosphorylation state. We previously identified DKK1 and GRB7 as genes with linked expression using Artificial Neural Network (ANN) analysis; here, we determine protein expression in a large cohort of early-stage breast cancer patients. Low levels of DARPP-32 Threonine-34 phosphorylation and DKK1 expression were significantly associated with poor patient prognosis, while low levels of GRB7 expression were linked to better survival outcomes. To gain insight into mechanisms underlying these associations, we analysed the transcriptome of T47D breast cancer cells following DARPP-32 knockdown. We identified 202 differentially expressed transcripts and observed that some overlapped with genes implicated in the ANN analysis, including PTK7, TRAF5, and KLK6, amongst others. Furthermore, we found that treatment of DARPP-32 knockdown cells with 17ß-estradiol or PKA inhibitor fragment (6-22) amide led to the differential expression of 193 and 181 transcripts respectively. These results underscore the importance of DARPP-32, a central molecular switch, and its downstream targets, DKK1 and GRB7 in breast cancer. The discovery of common genes identified by a combined patient/cell line transcriptomic approach provides insights into the molecular mechanisms underlying differential breast cancer prognosis and highlights potential targets for therapeutic intervention.

PP1, PKA and DARPP-32 in breast cancer: A retrospective assessment of protein and mRNA expression.

Cyclic AMP-dependent protein kinase A (PKA) and protein phosphatase 1 (PP1) are proteins involved in numerous essential signalling pathways that modulate physiological and pathological functions. Both PP1 and PKA can be inhibited by dopamine- and cAMP-regulated phosphoprotein 32 kD (DARPP-32). Using immunohistochemistry, PKA and PP1 expression was determined in a large primary breast tumour cohort to evaluate associations between clinical outcome and clinicopathological criteria (n > 1100). In addition, mRNA expression of PKA and PP1 subunits was assessed in the METABRIC data set (n = 1980). Low protein expression of PKA was significantly associated with adverse survival of breast cancer patients; interestingly, this relationship was stronger in ER-positive breast cancer patients. PP1 protein expression was not associated with patient survival. PKA and PP1 subunit mRNA was also assessed; PPP1CA, PRKACG and PRKAR1B were associated with breast cancer-specific survival. In patients with high expression of DARPP-32, low expression of PP1 was associated with adverse survival when compared to high expression in the same group. PKA expression and PP1 expression are of significant interest in cancer as they are involved in a wide array of cellular processes, and these data indicate PKA and PP1 may play an important role in patient outcome.

Dopamine and cAMP-regulated phosphoprotein 32kDa (DARPP-32), protein phosphatase-1 and cyclin-dependent kinase 5 expression in ovarian cancer.

Dopamine and cyclic-AMP activated phosphoprotein Mr32kDa (DARPP-32) is a central signalling protein in neurotransmission. Following DARPP-32 phosphorylation by protein kinase A (PKA), DARPP-32 becomes a potent protein phosphatase 1 (PP1) inhibitor. DARPP-32 can itself inhibit PKA following DARPP-32 phosphorylation by cyclin-dependent kinase 5 (Cdk5). Increasing evidence indicates a role for DARPP-32 and its associated signalling pathways in cancer; however, its role in ovarian cancer remains unclear. Using immunohistochemistry, expression of DARPP-32, PP1 and Cdk5 was determined in a large cohort of primary tumours from ovarian cancer patients (n = 428, 445 and 434 respectively) to evaluate associations between clinical outcome and clinicopathological criteria. Low cytoplasmic and nuclear DARPP-32 expression was associated with shorter patient overall survival and progression-free survival (P = .001, .001, .004 and .037 respectively). Low nuclear and cytoplasmic DARPP-32 expression remained significantly associated with overall survival in multivariate Cox regression (P = .045, hazard ratio (HR) = 0.734, 95% confidence interval (CI) = 0.542-0.993 and P = .001, HR = 0.494, 95% CI = 0.325-0.749, respectively). High cytoplasmic and nuclear PP1 expression was associated with shorter patient overall survival and high cytoplasmic PP1 expression with shorter progression-free survival (P = .005, .033, and .037, respectively). High Cdk5 expression was associated with shorter progression-free survival (P = .006). These data suggest a role for DARPP-32 and associated signalling kinases as prognostic markers with clinical utility in ovarian cancer.

Retrospective assessment of cyclin-dependent kinase 5 mRNA and protein expression and its association with patient survival in breast cancer.

Cyclin-dependent kinase 5 (Cdk5) is an atypical member of the cyclin-dependent kinase family and functions as a serine/threonine kinase that can be activated by non-cyclin binding activators p35 or p39. Cdk5 expression and activity has been linked with the development and progression of cancer; however, its expression in breast cancer has not been fully described. Protein expression of Cdk5 was determined in a large cohort of early-stage invasive breast cancer tumours (n = 1110) with long-term follow-up data using immunohistochemistry. Expression of CDK5 mRNA was assessed in the METABRIC cohort (n = 1980). Low nuclear and cytoplasmic expression of Cdk5 expression was significantly associated with shorter breast cancer-specific survival (P = .004 and P = .001, respectively). Importantly, low nuclear and cytoplasmic expression of Cdk5 remained associated with survival in multivariate analysis, including potentially confounding factors (hazard ratio (HR) = 0.612, 95% confidence interval (CI) = 0.418-0.896, P = .011 and HR = 0.507, 95% CI = 0.318-0.809, P = .004, respectively). In addition, low nuclear and cytoplasmic expression of Cdk5 was significantly associated with clinicopathological criteria associated with adverse patient prognosis. Low CDK5 mRNA expression was associated with shorter patient survival (P = .005) in the METABRIC cohort; no associations between copy gain or loss and survival were observed. These data suggest that low Cdk5 expression is associated with poor clinical outcome of breast cancer patients and may be of clinical relevance.