PO-286 Protein kinase D3: a crucial regulator of breast cancer stem cell survival

Abstract

IntroductionTriple negative breast cancer (TNBC) accounts for 15%–20% of all breast carcinomas in women. TNBC is particularly aggressive, highly metastatic and associated with poor prognosis. We previously identified Protein Kinase D3 (PKD3) to be upregulated in TNBC. PKD3 is a serine-threonine kinase that is best known for its functions in membrane trafficking and actin remodelling. Here we identify a novel role for PKD3 in breast cancer stem cell (CSC) maintenance. CSCs are endowed with tumor-initiating capacity, they possess increased chemoresistance and are known to initiate metastasis, and therefore are responsible for tumour relapse.Material and methodsIn the TNBC cell line MDA-MB-231 with a stable PKD3 knockdown, we performed a flow cytometry based screen of cell surface proteins, revealing the downregulation of several membrane proteins associated with CSCs. The importance of PKD3 for driving a CSC gene expression signature, maintaining CSC numbers and function was tested by qRT-PCR analysis of CSC markers, by measuring aldehyde dehydrogenase (ALDH) activity and performing mammosphere assays, respectively. Paclitaxel sensitivity of TNBC cell lines was assessed in combination with the selective PKD inhibitor CRT0066101. Whether the ectopic expression of PKD3 was sufficient to induce the expansion of the CSC pool was analysed in the non-transformed breast epithelial cell line MCF10A.Results and discussionsIn MDA-MB-231 cells with PKD3 knockdown, the CSC markers (CD184, Notch4, Sox2, Oct3/4) were downregulated. This was associated with reduced CSC numbers based on ALDH positivity and sphere-forming efficiency in mammosphere assays. Pharmacological PKD inhibition in the TNBC cell lines MDA-MB-231, BT549 and MDA-MB-436 verified these results. By contrast, inducible PKD3 expression in MCF10A cells elevated mammosphere formation and stem cell marker expression. Importantly, the combination of paclitaxel with CRT0066101 was superior over the single treatments in suppressing the survival of MDA-MB-231, BT549 and MDA-MB-436 cells in both colony formation and mammosphere assays.ConclusionOur results establish PKD3 as a crucial regulator of CSC maintenance and demonstrate that paclitaxel treatment in combination with PKD inhibition provides a potential novel strategy for future therapeutic applications in TNBC treatment.