Introduction: Active, phosphorylated AKT (pAKT) is involved in a diverse array of processes in breast cancer cells including proliferation, inhibition of apoptosis and therapeutic resistance. Ca²⁺ influx through specific channels has been linked to AKT phosphorylation in other cell types; however, this association has not been explored in breast cancer cells. HCC1569 is a HER2 positive breast cancer cell line, belonging to the basal molecular subtype. Breast cancers of the basal molecular subtype are associated with poor prognosis, hence identifying new drug targets, may lead to improved treatment of this breast cancer subtype

Aims: To develop a medium-throughput immunofluorescence assay for detection of pAKT in HCC1569 breast cancer cells and to identify Ca²⁺ channels or pumps that modulate basal pAKT levels in HCC1569 breast cancer cells.

Methods: A high content imaging screen was developed through single cell analysis of the integrated cytoplasmic fluorescence intensity of basal, epidermal growth factor (EGF) stimulated (positive control) and MK2206 (AKT inhibitor, negative control) treated HCC1569 cells. Once a robust medium-throughput (Z' score > 0) assay had been developed, an siRNA screen was performed by treating cells with siRNA targeted to different Ca²⁺ pumps, channels and channel modulators. Immunoblotting was performed as a confirmation assay.

Results: Optimisation of the developed pAKT assay led to an assay with a Z' score of 0.448, deemed suitable for a medium- throughput screen. Using this optimised assay, three proteins were identified as potentially lowering pAKT levels after silencing. Immunoblotting confirmed that TRPC1 siRNA-mediated silencing significantly reduced pAKT levels in HCC1569 breast cancer cells (p<0.05).

Discussion: Silencing of TRPC1 leads to a reduction of AKT phosphorylation in HCC1569 cells. Further studies are required to define the role of TRPC1 in the proliferation of HCC1569 breast cancer cells and their resistance to some inducers of apoptosis. Future studies should also assess the role of TRPC1 in other molecularly defined basal breast cancer cell lines.