Investigating the therapeutic potential of targeting NF-kappaB p52 in pre-clinical models of breast cancer.
Yeo, Syn Kok
mesheuropmc: parasitic diseases
Apoptosis is an important process in normal mammary gland physiology and has been implicated in mammary gland involution. In breast cancer cells, apoptotic resistance is an acquired feature that can promote tumour growth and progression. In order to assess the importance of apoptosis in these processes, caspases were directly inhibited by conditional expression of baculovirus p35 protein, a pan-caspase inhibitor, in the mammary glands of mice (rtTA/p35 mouse model). Inhibition of caspases during the first phase of involution in rtTA/p35 mice increased the number of sloughed luminal bodies which stained negatively for cleaved caspase-3. However, the total number of sloughed luminal bodies between mice with and without p35 expression was not changed. This suggests that caspase activation is not essential for the sloughing of mammary epithelial cells into lumen of alveoli and the initiation of involution may be a caspase-independent event. The importance of apoptotic resistance in breast cancer progression was also addressed by crossing rtTA/p35 mice with mice over-expressing the ErbB2 oncogene (Neu). Expression of p35 in established Neu mammary tumours did not affect the growth rates of tumours relative to un-induced controls. However, an increased number of mice with lung and liver metastases were observed when p35 was induced. This result substantiates the importance of apoptotic resistance in promoting metastasis and warrants the targeting of apoptosis regulators as an anti-metastatic therapy.\ud The NF-κB signalling pathway regulates a range of anti-apoptotic genes and can also induce the expression of a variety of metastatic promoting genes. Accordingly, the role and potential of the NF-κB p52 subunit as a therapeutic target was investigated. Firstly, this was addressed by silencing the Nfkb2 gene, which encodes p100/p52, in mammary cancer cell lines. Interestingly, assessment of these cells in in vitro assays measuring motility and tumour initiating potential revealed opposing effects in differing cell lines upon Nfkb2 knockdown. In N202.1A cells (ER-ve/ErbB2+ve), therapeutically beneficial effects were seen whereas in the 4T1 cell line (ER+ve/ErbB2-ve), malignancy was exacerbated. One explanation for the differential effects observed is that the negative regulatory subunit p100 and the transcriptional subunit p52 alternately have dominant roles in the respective cell lines. Although these results demonstrate a role for p52 in regulating tumour initiating potential in particular cell lines, the contextual outcomes indicate that targeting p52 at the gene level may be detrimental in cases where the loss of p100 outweighs the loss of p52. This indicates that strategies aimed at disrupting p52 activity should circumvent the loss of p100. Therefore, we addressed the possibility of diminishing p52 transcriptional activity via promotion of repressive transcriptional complexes. As the formation of repressive complexes is favoured by phosphorylation of Ser-222 in p52, we addressed the effects of over-expressing the S222D p52 mutant in 4T1 cells. In in vitro assays assessing proliferation, colony forming potential, tumour initiating potential and motility, we did not observe any differences upon expression of S222D p52. However, over-expression of S222A p52 did increase the motility of 4T1 cells and this demonstrates that the lack of Ser-222 phosphorylation (promoting transcriptionally active complexes) can contribute to the malignancy of breast cancer cells.\ud Due to the context dependent effects of Nfkb2 silencing, it was important to identify the particular subtypes of breast cancer which are dependent on p52 activity. We assessed a cohort of BRCA2-/- p53-/- tumours for nuclear p52 staining along with markers for respective breast cancer subtypes by immuno-histochemistry and found a positive correlation between p52 and p63. This suggests that aberrant p52 activity may be common in basal-like breast cancers which typically express p63 and could benefit most from p52 targeted therapies. In summary, our results indicate that targeting p52 in breast cancer cells can be therapeutically beneficial but only in particular subtypes of breast cancer and by certain therapeutic strategies.