Non-Canonical EZH2 Transcriptionally Activates RelB in Triple Negative Breast Cancer

<div><p>Enhancer of zeste homology 2 (EZH2) is the methyltransferase component of the polycomb repressive complex (PRC2) which represses gene transcription via histone H3 trimethylation at lysine 23 (H3K27me3). EZH2 activity has been linked with oncogenesis where it is thought to block expression of certain tumor suppressors. Relative to a role in cancer, EZH2 functions to promote self-renewal and has been shown to be important for the tumor-initiating cell (TIC) phenotype in breast cancer. Recently a non-canonical role for EZH2 has been identified where it promotes transcriptional activation of certain genes. Here we show that EZH2, through a methyltransferase-independent mechanism, promotes the transcriptional activation of the non-canonical NF-κB subunit RelB to drive self-renewal and the TIC phenotype of triple-negative breast cancer cells.</p></div

The HMT activity of EZH2 is not Required for RelB Transcription.

<p>(A) Immunoblots of indicated proteins in SUM-149 or MDA-MB-231 cells treated with the indicated concentrations of UNC1999 (left panel) or DZNep (right panel) for 72 hours. β-actin serves as a loading control. (B) Immunoblots of indicated proteins in SUM-149 or MDA-MB-231 cells expressing control siRNA or siRNA targeting EZH2, SUZ12 or both EZH2 and SUZ12. β-actin serves as a loading control. (C) ChIP analysis of EZH2 and SUZ12 binding at the RelB promoter or the MYT1 Promoter as a positive control in SUM-149 and MDA-MB-231 cells. Non-specific binding was estimated from IgG immunoprecipitates. Values given as means ± standard deviation from three technical repeats of one representative experiment from the three biological repeats.</p

Self-renewal of TICs in ER+ Breast Cancer Cells is not Dependent on EZH2 and RelB.

<p>(A) Top Panel, Quantification of tumorspheres formed by 100 MCF7, HCC-1428 or T47D cells expressing control siRNA or siRNA targeting EZH2 or RelB. Values given as means ± standard deviation from three biological repeats. *P<0.05 versus controls; two tailed unpaired t-test. Bottom Panel, Immunoblots of indicated proteins in adherent MCF7, HCC-1428 or T47D cells expressing indicted siRNA construct. β-actin serves as a loading control. (B) Left panel, quantification of tumorpsheres formed by 100 MCF7, HCC-1428 or T47D cells treated daily for 3 days with 1μM UNC1999. Values given as means ± standard deviation from three biological repeats. Right panel, immunoblots of the indicated proteins in adherent MCF7, HCC-1428 or T47D cells treated daily for 3 days with 1μM UNC1999 to validate EZH2 inhibition. β-actin serves as a loading control.</p

NF-κB DNA binding activity is reduced in IKKα and in IKKβ deficient cells.

<p>DNA binding activity of NF-κB was measured by gel shift assay. Indicated MEF cells were treated with TNFα for indicated times. Nuclear proteins were subject to gel shift assay for DNA binding analysis.</p

The role of IKKα and IKKβ in p65 phosphorylation and IκBα degradation in response to TNFα.

<p>MEF cells that are deficient for IKKα, IKKβ, or both IKKα and IKKβ (DKO) were treated with TNFα for the indicated times. NF-κB activity, as measured by IκBα degradation and p65 phosphorylation, is diminished in IKKα and IKKβ deficient MEF cells. IKKα and IKKβ DKO cells show no detectable p65 phosphorylation. Tubulin levels are shown as a loading control.</p

IKKα and IKKβ knock-down in Hela cells leads to diminished IκB degradation and p65 phosphorylation.

<p>HeLa cells were grown in 6-well plates and transfected with the indicated siRNA for 3 days. Western blots were performed on total cell extracts after treatment with TNF for the indicated times. Tubulin levels are shown as a loading control.</p

IKKβ kinase mutant inhibits TNF and IKKα-induced NF-κB-dependent reporter gene activity.

<p>WT and IKKβ null MEFS were transfected with the indicated vector construct and with the NF-κB-dependent luciferase reporter. Luciferase luciferase activity was measured 48 hr after transfected. Where indicated, cells were treated with TNF for 4 hrs. Relative luciferase values were calculated using a renilla control expression vector for normalization. Relative luciferase values are normalized to vector control samples.</p

IKKα and IKKβ each contribute to TNF-induced NF-κB activity in HeLa cells.

<p>HeLa cells, seeded in 24-well plates were transiently transfected with indicated siRNA constructs for 48 hr. Then media was replaced and cells were further transfected with NF-κB responsive 3x-κB luciferase and a control Renilla luciferase contructs. TNF was added (as indicated) and 24 hr later cells were lysed and dual lucifearse assay was performed. Luciferase readings in untreated and control vector transfected cells were normalized as 1.</p

Activity comparison for TBK1 and IKKε.

<p>Number of active compounds (N) detected in each screen for total number detected (unfiltered), number after drug like filtering (filtered.drug-like), hits from the LOPAC and Kinase libraries, and the number of chemical clusters and singleton hits as described in the text.</p

Identification of the optimal phosphorylation motif for TBK1.

<p>A-B) The positional scanning peptide library technology was used to determine the optimal phosphorylation motif for recombinant A) GST-TBK1 WT or B) kinase-dead GST-TBK1 K38A as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041494#pone.0041494-Hutti3" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041494#pone.0041494-Turk1" target="_blank">[28]</a>. Briefly, 198 peptide libraries were phosphorylated in individual kinase assays. The sequence for these libraries is Y-A-X-X-X-Z-X-S/T-X-X-X-X-A-G-K-K-biotin (Z =  fixed amino acid, X =  equimolar mixture of amino acids excluding Ser, Thr, and Cys). After binding to a streptavidin-coated membrane, phosphorylation was visualized by the incorporation of ³²P. C) Primary and secondary selections for TBK1, as determined in A). D) 50 µM of the indicated peptide was phosphorylated in an <i>in vitro</i> kinase assay with recombinant GST-TBK1 for 30 min. Phosphorylation of each peptide is shown as a percentage of the rate of phosphorylation of TBK1-Tide, the optimal peptide substrate for TBK1. Error bars are standard deviation.</p