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Opposing roles of Nfkb2 gene products p100 and p52 in the regulation of breast cancer stem cells

Breast Cancer Research and Treatment volume 162, pages 465–477 (2017)Cite this article

Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusion

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.

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Acknowledgement

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.

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Authors and Affiliations

  1. Department of Cancer Biology, University of Cincinnati, Vontz Building, 3125 Eden Avenue, Cincinnati, OH, 45267, USA

    Syn Kok Yeo

  2. European Cancer Stem Cell Research Institute, School of Biosciences, Cardiff University, Hadyn Ellis Building, Maindy Road, Cathays, Cardiff, CF24 4HQ, UK

    Rhiannon French, Filomena Spada & Richard Clarkson

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  1. Syn Kok Yeo
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  2. Rhiannon French
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Correspondence to Syn Kok Yeo.

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Conflict of interest

Authors declare no conflict of interest.

Ethical standards

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.

Electronic supplementary material

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10549_2017_4149_MOESM1_ESM.tif

Silencing of Nfkb2 does not affect the proliferation and colony-forming potential of mammary cancer cell lines. a Immunoblot showing the levels of p100 and p52 in 4T1 and N202.1A cells after transduction with Non-Target (NT) or Nfkb2 shRNA. Growth curves showing the number of cells over time for (b) 4T1 and (c) N202.1A cells. d Colony-forming assay of 4T1 and N202.1A cells seeded in 6-well plates at a density of 1000 cells/well and quantified after 7 days. Data points represent average of at least n = 6 and error bars indicate standard error of the mean (SEM). N.S. indicates a non-significant difference, where statistical significance relative to NT controls was determined by two-tailed t-test. Supplementary material 1 (TIFF 70 kb)

10549_2017_4149_MOESM2_ESM.tif

Silencing of Nfkb2 with independent shRNAs in 4T1 cells. a Immunoblot showing levels of p100 and p52 after knockdown with non-target, p52 sh43 or p52 sh44 in 4T1 cells. b Bar chart showing number of migrated cells in a 10 × field of view for Boyden chamber migration assays of 4T1 non-target, p52 sh43 or p52 sh44 cells. c Bar chart showing number of spheres formed for mammosphere-forming assays of 4T1 non-target, p52 sh43 or p52 sh44 cells. Supplementary material 2 (TIFF 66 kb)

10549_2017_4149_MOESM3_ESM.tif

Effects of NMS-873 treatment on CSC marker expression in human breast cancer cell lines. a Representative dot plots showing expression of CD44 + and CD24 + in MDA-MB-231 and SKBR3 cell lines treated with vehicle or 1 μM NMS-873 for 48 h under normal culture conditions and analysed by flow cytometry. Bar charts showing the percentage of b CD44+CD24−, c CD44+CD24+, or d CD44−CD24+ populations. Data points represent average of at least n = 6 and error bars indicate standard error of the mean (SEM), **indicates p < 0.01, where statistical significance relative to vehicle controls was determined by two-tailed t-test. Supplementary material 3 (TIFF 116 kb)

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Yeo, S.K., French, R., Spada, F. et al. Opposing roles of Nfkb2 gene products p100 and p52 in the regulation of breast cancer stem cells. Breast Cancer Res Treat 162, 465–477 (2017). https://doi.org/10.1007/s10549-017-4149-0

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Keywords

  • Breast cancer
  • Nfkb2
  • NF-KappaB
  • Cancer stem cell
  • ErbB2
  • p97 ATPase