PKCθ promotes c-Rel–driven mammary tumorigenesis in mice and humans by repressing estrogen receptor α synthesis
Karine Belguise, Gail E. Sonenshein
Karine Belguise, Gail E. Sonenshein
Published December 3, 2007
Citation Information: J Clin Invest. 2007;117(12):4009-4021. https://doi.org/10.1172/JCI32424.
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Research Article Oncology

PKCθ promotes c-Rel–driven mammary tumorigenesis in mice and humans by repressing estrogen receptor α synthesis

Abstract

The vast majority of primary human breast cancer tissues display aberrant nuclear NF-κB c-Rel expression. A causal role for c-Rel in mammary tumorigenesis has been demonstrated using a c-Rel transgenic mouse model; however, tumors developed with a long latency, suggesting a second event is needed to trigger tumorigenesis. Here we show that c-Rel activity in the mammary gland is repressed by estrogen receptor α (ERα) signaling, and we identify an epigenetic mechanism in breast cancer mediated by activation of what we believe is a novel PKCθ-Akt pathway that leads to downregulation of ERα synthesis and derepression of c-Rel. ERα levels were lower in c-Rel–induced mammary tumors compared with normal mammary gland tissue. PKCθ induced c-Rel activity and target gene expression and promoted growth of c-Rel- and c-RelxCK2α–driven mouse mammary tumor–derived cell lines. RNA expression levels of PKCθ and c-Rel target genes were inversely correlated with ERα levels in human breast cancer specimens. PKCθ activated Akt, thereby inactivating forkhead box O protein 3a (FOXO3a) and leading to decreased synthesis of its target genes, ERα and p27Kip1. Thus we have shown that activation of PKCθ inhibits the FOXO3a/ERα/p27Kip1 axis that normally maintains an epithelial cell phenotype and induces c-Rel target genes, thereby promoting proliferation, survival, and more invasive breast cancer.

Authors

Karine Belguise, Gail E. Sonenshein

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Figure 3

Inhibition of PKCθ with rottlerin reduces binding and transactivation by c-Rel.

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Inhibition of PKCθ with rottlerin reduces binding and transactivation by...
(A) WCEs (500 μg) prepared from MMTV–c-Rel mouse mammary tumors (19-month-old mice) and normal tissues (2-month-old mice, except 4948-N 19-month-old mice), were subjected to a PKC kinase assay (top). As controls for PKC kinase specificity, WCE from 4528-T was incubated with either normal IgG and the kinase reaction performed as described above or with PKC antibody, and the kinase reaction was performed in the presence of 25 nM staurosporine. Top: Samples were resolved on 1 gel (left panel) and on 2 gels (3 right panels). The 2 far-right panels were from 1 gel; these panels have been merged with the second gel to compare tumor and normal samples. Lanes were cut to align the sample order. Middle and bottom: WCEs used for the PKC kinase assay were run on 3 gels and subjected to immunoblotting for PKC and β-actin. Lanes were cut to align the sample order. WCEs from mammary glands of control wild-type FVB mice or transgenic animals (4948-N) (at 19 months of age) were similarly processed. (B) rel-3983 and rel/CK2-5839 cells were treated with 25 nM staurosporine (left), 5 μM rottlerin (middle), 1 μM Gö6983 (right), or DMSO for 20 hours, and WCEs (500 μg) were subjected to ONP assay, as described in Figure 1D. The input and the oligonucleotide precipitated proteins were run on the same gel but were noncontiguous. (C) WCEs (250 μg) prepared from the indicated mouse mammary tumor or normal tissue were subjected to a PKCθ kinase assay (62). As a negative control, WCE from 4556-T was incubated with normal IgG and similarly processed. (D) rel-3983, rel-3875, and rel/CK2-5839 cells were transiently transfected in triplicate with 0.05 μg pG5E1B-Luc vector, β-gal, or 0.1 μg Gal4 or Gal4–c-Rel vectors as indicated. Sixteen hours later, cells were treated with 5 μM rottlerin or DMSO for 20 hours and processed as described in Figure 2D.