A Transgenic Model Reveals Important Roles for the NF-κB Alternative Pathway (p100/p52) in Mammary Development and Links to Tumorigenesis

Abstract

A regulated pattern of nuclear factor B (NF-B) activation is essential for normal development of the mammary gland. An increase in NF-B activity has been implicated in breast cancer. We have generated a novel transgenic mouse model to investigate the role of the alternative NF-B pathway in ductal development and identify possible mediators of tumorigenesis downstream of p100/p52. By overexpressing the NF-B p100/p52 subunit in mammary epithelium using the ␤-lactoglobulin milk protein promoter, we found that transgene expression resulted in increased overall NF-B activity during late pregnancy. During pregnancy, p100/p52 expression resulted in delayed ductal development with impaired secondary branching and increased levels of Cyclin D1, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and cyclo-oxygenase-2 (COX-2) in the mammary gland. After multiple pregnancies the p100 transgenics exhibited a ductal thickening accompanied by small hyperplastic foci. In tumors from mice expressing the polyoma middle T oncoprotein (PyVT) in the mammary gland, increased levels of p100/p52 were present at the time of tumor development. These results show that increased p100/p52 disrupts normal ductal development and provides insight into the mechanism by which this may contribute to human breast cancer.The development of the murine mammary gland is a complex process. A simple ductal structure forms during embryogenesis through signaling between the epithelium and surrounding mesenchyme. The cyclic production of hormones promotes further ductal growth and branching during puberty. The majority of mammary gland proliferation is observed in pregnancy, with ductal branching and the formation of secretary lobular alveolar units. Following lactation and the weaning of pups there is a re-modeling of the epithelium termed involution, and the gland returns to the simpler structure observed in virgin animals (1-3).The transcription factor nuclear factor B (NF-B) 2 is involved in the process of mammary development. NF-B is a dimer formed from a multisubunit family consisting of p65 (Rel A), Rel B, c-Rel, p105/p50 (NF-B1), and p100/p52 (NF-B2; Ref. 4). The activation of NF-B is regulated by the binding of inhibitory proteins termed IBs (5). The C-terminals of the p105 and p100 subunits share homology with IB (6, 7). These inhibitory subunits are phosphorylated in response to stimuli by an upstream IB kinase complex (IKK). This contains two kinases, IKK␣ and IKK␤, and a regulatory subunit, IKK␥ (NEMO; Refs. 8 -10). In the canonical NF-B pathway, a dimer composed of the p65 and p50 subunits is held in the cytosol through binding of the inhibitor IB␣. The phosphorylation of IB␣ by IKK␤ acts as a signal for the ubiquitination and degradation of the inhibitor (11). This allows p50/p65 to translocate to the nucleus and bind to the promoter of target genes that are involved in a diverse range of processes including inflammation, cell growth, and apoptosis (4, 12). More recently an alternative pathway of NF-B subunit activation ...

NF‐κB and epithelial to mesenchymal transition of cancer

“…Consistent with the above data, transgenic mice overexpressing NF-kB p100/p52 under control of the b-lactoglobulin promoter display elevated MMP-9 and MMP-2 activity in the mammary gland [Connelly et al, 2007]. Although the mammary glands did not appear to undergo EMT, increased hyperplastic growth of mammary cells was found [Connelly et al, 2007]. Lastly, expression of a constitutively active IKKe/i in kidney epithelial cells caused a significant increase in MMP-9 expression [Boehm et al, 2007], consistent with its role in invasive phenotype [Eddy et al, 2005].…”

“…Thus, NF-kB indirectly regulates MMP-2 activity, which is distinct from the direct transcriptional regulation seen for MMP-9. Consistent with the above data, transgenic mice overexpressing NF-kB p100/p52 under control of the b-lactoglobulin promoter display elevated MMP-9 and MMP-2 activity in the mammary gland [Connelly et al, 2007]. Although the mammary glands did not appear to undergo EMT, increased hyperplastic growth of mammary cells was found [Connelly et al, 2007].…”

IKK1 and IKK2 cooperate to maintain bile duct integrity in the liver

“…Although at this stage it is not clear why the combined ablation of IKK1 and IKK2 or IKK1 and NEMO does not result in HCC development, it is likely that the additional ablation of IKK1 interferes with liver cancer development in this model. Although the mechanisms by which IKK1 might control liver tumorigenesis in NEMO LPC-KO mice remain elusive, recent studies implicated IKK1 and the alternative NF-B pathway in oncogenesis (27)(28)(29)(30)(31). In the mammary gland, IKK1, p100/p52, and relB were shown to regulate cyclin D1 expression and epithelial cell proliferation, and mice lacking inducible IKK1 kinase activity showed delayed development of ErbB2-induced tumors, implicating alternative NF-B signaling in mammary epithelial hyperplasia and oncogenesis (27,29,31).…”

“…Although the mechanisms by which IKK1 might control liver tumorigenesis in NEMO LPC-KO mice remain elusive, recent studies implicated IKK1 and the alternative NF-B pathway in oncogenesis (27)(28)(29)(30)(31). In the mammary gland, IKK1, p100/p52, and relB were shown to regulate cyclin D1 expression and epithelial cell proliferation, and mice lacking inducible IKK1 kinase activity showed delayed development of ErbB2-induced tumors, implicating alternative NF-B signaling in mammary epithelial hyperplasia and oncogenesis (27,29,31). In addition, IKK1 was recently shown to antagonize p53-mediated gene transcription by mediating phosphorylation of CBP and switching its preference toward NF-B-induced gene expression promoting cell growth (28).…”

High IKKα expression is associated with reduced time to recurrence and cancer specific survival in oestrogen receptor (ER)‐positive breast cancer

“…Oestrogen inhibits the Notch pathway and application of anti-oestrogens result in the activation of Notch 13 and the kinase activity of IKKα has been found to be associated with Notch in the activation of ER-dependent genes 14 . In mice a delay in mammary gland development is observed when p100/52 is overexpressed and these mice developed multiple tumours 15 , however other information is lacking. The current study aims to assess if members of the non-canonical…”