EZH2 is a transcriptional activator of RelB.

(A) Immunoblots of indicated proteins in SUM-149, MDA-MB-231 or BT-549 cells expressing control siRNA or siRNA targeting p65, RelB or EZH2. β-actin serves as a loading control. (B) Real time PCR showing expression of RelB in SUM-149, MDA-MB-231 and BT-549 cells. Expression was normalized to glucuronidase beta. Values given as means ± standard deviation from three biological repeats. (C) Dual luciferase assay from HEK293 cells co-expressing the RelB Reporter with either Flag-p65 or HA-EZH2 and Renilla luciferase. Relative light units (RLU) was normalized to Renilla luciferase light units and expressed as fold over the RelB promoter alone. Values given as means ± standard deviation of four biological repeats. *P = 0.05, **P< 0.05 versus controls; two tailed unpaired t-test. (D) ChIP analysis of p65 and EZH2 binding at the RelB promoter 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

EZH2 and RelB are Required for the Maintenance of TNBC TIC.

(A) Top panel, Quantification of tumorspheres formed by 100 SUM-149 or MDA-MB-231 cells expressing control siRNA or siRNA targeting EZH2 or RelB. Values given as means ± standard deviation from three biological repeats. *P<0.05, **P< 0.005 versus controls; two tailed unpaired t-test. Bottom panel, Immunoblots of indicated proteins in adherent SUM-149 or MDA-MB-231 cells expressing the indicated siRNA construct. β-actin serves as a loading control. (B) Quantification of tumorspheres formed by either 100 SUM-149 or MDA-MB-231 cells treated daily for 3 days with 1μM UNC1999. Values given as means ± standard deviation from three biological repeats. **P< 0.005 versus controls; two tailed unpaired t-test.</p

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

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