PKCζ regulates cell polarisation and proliferation restriction during mammary acinus formation

Abstract

SummaryMammary epithelial cells organize in three dimensions and generate acini when supported on laminin-rich extracellular matrix. Acinus formation begins with the apicobasal polarisation of the outer cells of the assembly and the withdrawal of these cells from the cell cycle. Internal cells then clear out to form a hollow lumen. Here, we show that PKC is phosphorylated (at T410) and activated in the early stages of acinus formation in both primary cells and MCF10A cells, and during mammary tree maturation in vivo. Phospho-PKC colocalised with tight junction components and bound to the Par polarising complex in developing acini. To further investigate the importance of PKC phosphorylation in this context, acinus formation was studied in MCF10A cells overexpressing nonphosphorylatable (T410A) or 'constitutively phosphorylated' (T410E) PKC. In both cell types, acinus-associated cell polarisation and lumen clearance were compromised, emphasising the importance of regulated phosphorylation of PKC at T410 for successful acinus formation. PKC can be activated in a phosphorylation (at T410)-dependent and a phosphorylation-independent manner. Cells overexpressing a complete kinase-deficient PKC (K281W) displayed a cell polarising deficit, but also generated large 'multi-acinar' structures with associated early lumenal cell hyperproliferation. Therefore our data shows, for the first time, that two separable PKC activities (one phosphorylation-dependent, the other not) are required to support the cell polarisation and proliferation restriction that underpins successful acinus formation. Paralleling these contributions, we found that low levels of PKC mRNA expression are associated with more 'poorly differentiated' tumours and a poor outcome in a cohort of 295 breast cancer patients.

The Rho GTPase Cdc42 is required for primary mammary epithelial cell morphogenesis in vitro

“…However, the reduction culture. 26 Taken together, these studies suggest that Cdc42 is crucial for polarity establishment and proper cell-cell adhesion in a variety of cell and tissue types.…”

“…24,25 Inhibition of PKCζ in primary MECs in 3D culture caused failure of the cells to mature into polarized acini. 26 We therefore, examined the effects of Cdc42 deficiency on cell polarity establishment and tight junction formation. For these experiments, the acini were stained with antibodies against polarity markers α6-integrin (basal), phospho-ERM (pERM) and PKCζ (apical), and tight junction marker zonula occluden-1 (ZO-1).…”

The MEF2–HDAC axis controls proliferation of mammary epithelial cells and acini formation in vitro

“…Human normal immortalized epithelial breast cells (MCF10A) (Whyte et al, 2010) and MCF10A cells expressing HER2 were maintained in Ham's F12: Dulbecco's modified Eagle's medium (DMEM) 1:1 medium (SigmaAldrich) supplemented with 5% horse serum (Gibco), penicillin (100 U/ ml), streptomycin (100 µg/ml), L-glutamine (2 mM) (Lonza), insulin (0.01 mg/ml), hydrocortisone (500 ng/ml), cholera toxin (100 ng/ml) (Sigma-Aldrich) and epithelial growth factor (20 ng/ml) (Peprotech). HEK-293T cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ ml) (Lonza).…”

c-Jun N-terminal kinase activity supports multiple phases of 3D-mammary epithelial acinus formation

“…These experiments reiterated the requirement for JNK signalling in driving cell polarisation during acinus formation (see also (Whyte et al, 2010)). A likely target of this (glucocorticoid) -JNK signalling axis is maintenance of expression of adhesion proteins (Murtagh et al, 2004), which play a role in establishing and maintaining the polarised state of organised epithelial cell aggregates.…”

“…Thus, there may be two cellular targets for JNK in this context. The first, which supports establishment of cell polarisation, probably lies upstream of PKCζ activation on a signalling path downstream of activated b1 integrin (Whyte et al, 2010). The later target, which supports lumen clearance, is likely to centre on rac1 activation (Keely et al, 1997;Muthuswamy et al, 2001).…”