Publication

Neurotrophic signalling and inhibition in breast cancer and chronic lymphocytic leukaemia

Hillis, Jennifer
Citation
Abstract
Neurotrophins are important growth factors for neuronal cells. They interact with the Trk kinase receptors and the p75NTR receptor to induce several signaling pathways. Neurotrophins and their receptors are known regulators of cell proliferation, survival and death in numerous cell types, in addition to neuronal cells. They are dysregulated in several diseases, including cancer. In particular, nerve growth factor (NGF) is frequently overexpressed in breast cancer cells, where TrkA and p75NTR signaling lead to ERK-dependent cell proliferation and NF-κB–dependent survival, respectively. In B cell malignancies expression of neurotrophins and their receptors is often increased on cellular stress, and may be involved in drug resistance. The first aim of my research was to investigate NF-κB regulation and signalling due to NGF/p75NTR in breast cancer. The ER positive cell line T47D was found to be unresponsive to NGF, so the focus moved to the triple negative type cell line MDA-MB-231. A small amount of I-κB phosphorylation was seen after NGF addition. Cells were transduced with an NF-κB luciferase reporter gene. These NF-κB reporter cells were used to look at the effect of NGF, NGF inhibition, p75NTR inhibition, and TrkA inhibition. As there was little effect of NGF addition and a significant decrease in NF-κB activity was seen on NGF signalling inhibition, it was hypothesised that these cells have constitutively high NF-κB activity. This reporter system was further used to test the relationship between other stress responses and NF-κB activity in breast cancer. Previous work had identified the NGF-binding inhibitor Ro 08-2750 as an inducer of cell death in chronic lymphocytic leukaemia (CLL) cells. My second aim was to examine the effect of neurotrophin and neurotrophin receptor inhibitors on CLL cells. On examination of neurotrophin and receptor expression in Mec1 cells, only p75NTR and TrkB95 were seen. Use of Tat-pep5, to inhibit p75NTR signalling, and K252a, to inhibit auto-phosphorylation of Trk receptors, resulted in cell death in Mec1 and primary CLL cells. This cell death was characterised, revealing little to no caspase activity, caspase cleavage or PARP cleavage. On RIPK1 inhibition cell death was partly reduced, suggesting possible necroptosis. This cell death was revealed to be ferroptosis, an emerging mode of cell death, due to its prevention by the ferroptosis inhibitor ferrostatin-1 and the requirement for functioning iron metabolism. This highlights the reduced capacity of CLL cells to undergo the conventional modes of cell death, and how alternative cell death pathways are potential targets for new cancer therapies. Overall, this thesis questions the specific role of p75NTR in promotion of NF-κB in breast cancer cells and describes the induction of ferroptosis in a CLL cell line for the first time.
Publisher
Publisher DOI
Rights
Attribution-NonCommercial-NoDerivs 3.0 Ireland