Opposing roles of Nfkb2 gene products p100 and p52 in the regulation of breast cancer stem cells
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Nuclear factor-kappa B (NF-κB) signalling has been shown to regulate properties of breast cancer stem cells. However, the specific contribution of the non-canonical NF-κB pathway, components of which are elevated in aggressive breast cancer has not been addressed.
Through shRNA silencing of the Nfkb2 gene, the role of p100/p52 in 4T1 and N202.1A cell lines were assessed by NF-κB reporter, invasion, tumoursphere and orthotopic transplantation assays. The processing of p100 into p52 was also inhibited with a p97 ATPase inhibitor, NMS-873, and its effects on tumoursphere formation was assessed.
Knockdown of Nfkb2 led to opposing changes in NF-κB-dependent transcription. NF-κB activity was elevated in 4T1 cells and this resulted in increased motility, cancer stem cell (CSC) activity and tumourigenicity in vivo. Conversely, depletion of Nfkb2 in N202.1a cells decreased NF-κB activity, CSC properties and tumourigenicity in vivo. By selectively overexpressing the p52 subunit in Nfkb2 depleted cells, we found that the increased malignancy in 4T1 cells could not be reverted in the presence of p52, whereas the decreased tumourigenicity of N202.1a cells could be rescued by p52. These results indicate that p100 and its subunit p52 have opposing effects on breast CSC activity. Accordingly, inhibition of an upstream regulator of p100 processing was effective in reducing tumoursphere formation of N202.1A and SKBR3 (ErbB2 HIGH) cells without aggravating that of 4T1 and MDA-MB-231 (ErbB2LOW) cells.
These findings indicate that inhibiting the processing of p100 may be a potential therapeutic strategy to suppress CSC activity in a subset of breast tumours.
KeywordsBreast cancer Nfkb2 NF-KappaB Cancer stem cell ErbB2 p97 ATPase
We are grateful to Derek Scarborough from the Histology Unit for assistance with histology and processing of tissues and the animal research facility at Cardiff University. We also appreciate the kind feedback and assistance given by Nader Omidvar, Alison Wakefield, Luke Piggott and Joseph Farmer. This work was supported by a Breast Cancer Campaign PhD studentship Grant (2007NovPhD06) awarded to R. Clarkson. We would also like to acknowledge the contribution of specimen donors and research groups who have contributed to the TCGA Breast dataset.
Compliance with ethical standards
Conflict of interest
Authors declare no conflict of interest.
The authors declare that the experiments performed in the current publication comply with the current laws of the United Kingdom and all maintenance, breeding and scientific procedures involving animals were carried out according to the guidelines set by the U.K. Home Office Regulations Animals (Scientific Procedures) Act 1986.
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