The role of IKKalpha and IKKalpha phosphorylation in breast cancer

Breast cancer is the most frequently diagnosed cancer in women in the UK. Although several targeted therapies have been approved for patients with oestrogen-positive and HER2-positive breast cancer, chemotherapy remains the standard systemic option for patients with triple-negative breast cancer (TNBC). The molecular mechanism driving tumour heterogeneity of TNBC has not been fully elucidated. Understanding molecular pathways and identifying biomarkers allow the discovery of the potential therapeutic targets for the treatment of this disease. The NF-κB pathway is a crucial link between inflammation and cancer. IKKα is an essential kinase of the pathway demonstrated to be involved in breast cancer progression through the NF-kB-dependent and NF-κB-independent functions. The current study aimed to investigate the role of IKKα, its phosphorylation at serine 176 (pIKKα-Ser176) and threonine 23 (pIKKα-Thr23), and their upstream kinases, NIK and pAKT in different breast cancer subtypes. Since it has been suggested that crosstalk exists between IKKα and androgen receptor (AR) in TNBC, expression of AR was also investigated. Immunohistochemistry was employed on a tissue microarray (TMA) of patients with breast cancer to assess the expression of IKKα, pIKKα-Ser176, pIKKα-Thr23, pAKT, NIK and AR. Overall, the findings reported here suggest that the prognostic role of these potential biomarkers differed among breasts cancer subtypes and differed depending on their cellular locations. Inconsistent results between the cohort were also observed. Several limitations need to be noted, including using the IHC-based classification instead of gene expression subtyping, which might affect the outcome. Using old TMAs was another limitation that might affect protein expressions. Using the multiple cut-off values to classify tumours with low and high expression of each protein might lead to an inconsistent result among the cohorts. Further study is needed, using gene expression subtyping, freshly cut TMAs and an appropriated method for determining a cut-off value. Another aim of the present study was to investigate differentially expressed gene profiles for tumours with low versus high cytoplasmic IKKα, cytoplasmic pIKKα-Ser176 and nuclear pIKKα-Thr23 in ER-negative tumours. We hypothesise that such DEGs are associated with distinct biological functions, representing a common mechanism of tumours with high IKKa, pIKKα-Ser176 and pIKKα-Thr23. 14 and 3 significant DEGs were observed in the IKKα and the pIKKα-Thr23 group. None was found in the pIKKα-Ser176 group. Several genes in the IKKα group have been reported to involve in breast cancer progression, including ACTA2, ALCAM, PDCD6IP, CD9, MVP, and PRSS8 (1-6). As the DEGs analysis was based on small sample size, the present study did not confirm the hypothesis regarding DEGs between tumours with high and low expression of the proteins of interest. The study should be repeated using an adequate number of samples. The impact of pharmacologically inhibiting IKKα in two ER-negative breast cancer cells with differing AR status was investigated. Inhibition on p52 processing was assessed by western blotting and phenotypic response was assessed by changes in cell viability. The impact of pharmacologically inhibiting IKKα in two ER-negative breast cancer cells with differing AR statuses was investigated. Inhibition on p52 processing was assessed by western blotting, and the phenotypic response was assessed by changes in cell viability. Using the western blot, the present study was unable to demonstrate an inhibitory effect on p52 by using first-in-class IKKα inhibitors. Future work is needed to repeat and perform this experiment in triplicate, which was not possible due to the COVID-19 restrictions. A dose dependent decrease in cell viability was observed after using the IKKα inhibitor. However, AR antagonist alone was sufficiency in ER-negative/AR-positive cell lines. In conclusion, the result in this thesis using three patients tissue cohorts suggest that IKKα, pIKKα-Ser176 and pIKKα-Thr23 may be associated with clinical outcome. However, the prognostic roles of these proteins are different depending on tumour subtypes and cellular locations, and no clear conclusion could be reached. There has been growing recognition of the crucial links between the NF-κB pathway and the critical components in the tumour microenvironment (TME). Future work investigating the complex linkage between the NF-κB pathway and the crucial components in TME in breast cancer, such as programmed death-ligand (PD-L1) and T-lymphocyte-associated protein 4 CTLA-4, is needed. This will serve as a therapeutic potential of inhibition either NF-κB or molecules involved in NF-κB activation such as IKKα inhibitors, combined with immune checkpoint blockade for patients with breast cancer, especially TNBC