The role of IKKalpha, IKKbeta and NF-kappaB in the progression of breast cancer

Breast cancer is the most common female cancer in the UK and, despite earlier detection and improved treatments, remains the second most common cause of cancer death in women. Although therapies exist for breast cancer, including endocrine therapy for oestrogen receptor (ER) positive tumours, resistance to current treatment remains a major problem. The molecular mechanisms of endocrine resistance have yet to be fully elucidated and in order to improve treatment for patients this needs to be addressed. Clinically breast cancer presents as several distinct diseases with different outcomes and molecular profiles. Over the past decade, through the use of molecular profiling, the number of different subtypes of breast cancer has grown and understanding the pathways driving each subtype may allow a stratified approach to therapy, allowing patients to receive the treatment which will be of most benefit. The Nuclear Factor kappa B (NF-κB) pathways regulate the transcription of a wide range of genes involved in the immune response, inflammation, proliferation and apoptosis. Many of these processes are hallmarks of cancer and NF-κB has been hypothesised to have a role in tumorigenesis. The aim of the current study was to investigate the role of both NF-κB pathways in the pathogenesis and recurrence of breast cancer. Immunohistochemistry was employed to assess key components of the canonical and non-canonical NF-κB pathways on a tissue microarray (TMA) of 544 patients with full clinical follow up and clinical information including ER status, subtype, necrosis, apoptosis and angiogenesis. Nuclear expression of p65 phosphorylated at serine 536 was associated with angiogenesis and shorter recurrence free interval. Cytoplasmic expression of IKKα was associated with cell death (apoptosis and necrosis) and a shorter recurrence free interval was also observed for those with high expression. These observations between phospho-p65/IKKα and recurrence free interval, when subdivided by ER status, remained significant in ER positive tumours but were negated in ER negative tumours. When split further into subtype, a diverging role for each was observed with phospho-p65 associating with recurrence in luminal B tumours and IKKα with luminal A tumours. Other members of the NF-κB pathways (p65, IKKβ, NIK and RelB) were not associated with recurrence free interval. When these results were tested in an independent cohort, IKKα remained significant on recurrence free interval and breast cancer specific survival in ER positive tumours however phospho-p65 was only marginally associated with breast cancer specific survival. Variability of phospho-p65 is a major issue in IHC studies and therefore an alternative marker of the canonical NF-κB pathway is required. Analysis of expression in this second cohort also revealed that high levels of IKKα in the cytoplasm were associated with recurrence on tamoxifen. This marker may therefore be able to be employed as a diagnostic tool to predict patients who are likely to display endocrine resistance and may represent a therapeutic strategy in combination with endocrine therapy, or for patients after endocrine resistance has occurred. Further examination of the pathways in breast cancer cell lines also demonstrated a difference between ER positive and ER negative breast cancer. In ER negative MDA-MB-231 cells phosphorylation of p65 (from the canonical NF-κB pathway) and phosphorylation of p100 (from the non-canonical NF-κB pathway) was apparent even in untreated control cells, suggesting constitutive activation. Expression was however found to be inducible in ER positive MCF7 cells. In order to investigate whether kinases involved in activation of each pathway, IKKβ in the canonical pathway and IKKα in the non-canonical NF-κB pathway, had potential as targets in breast cancer, we examined the phenotypic impact of silencing their expression in breast cancer cell lines. Silencing IKKβ induced apoptosis and decreased cell viability in both MCF7 and MDA-MB-231 cells but reduction in expression of IKKα only impacted on cell viability and apoptosis in ER positive MCF7 cells. This data, consistent with results from the clinical specimens, has therefore revealed that inhibitors of IKKα are likely to be most beneficial in the treatment of ER positive tumours. These results suggest that the NF-κB pathways are associated with recurrence in patients with ER positive tumours with each pathway possibly associating with recurrence in different subtypes. Additional studies in a larger cohort, including patients receiving aromatase inhibitors are required, accompanied by extensive mechanistic studies to further explore the roles of IKKα and IKKβ in breast cancer. These observations highlight that different subgroups of breast cancer may have different signalling pathways driving progression and therefore patients are likely to benefit from different therapeutic strategies.