When cells are deprived of E2, DAXX expression decreases in a time-dependent manner to almost undetectable by day 2 (Fig. DAXX-inducing phytoestrogens were tested to assess selectivity towards ER and/or ER. Results showed that phytoestrogens tested induced DAXX protein expression and inhibited survival of TICs from ER+ MCF-7 and T47D cells. Only naringenin, resveratrol, and quercetin did not stimulate total cell proliferation. Naringenin, resveratrol, but not quercetin inhibited survival of TICs in vitro and in vivo in a DAXX-dependent manner. Naringenin-induced DAXX protein expression and inhibition of TICs seemed to Akt1 be more selective towards ER while resveratrol was more selective through ER. Naringenin or resveratrol inhibited the rate of tumor initiation and rate of tumor growth in a DAXX-dependent manner. These results suggest that a therapeutic approach using a phytoestrogen to induce DAXX protein expression could potently inhibit TICs within a tumor to delay or prevent tumor initiation. Therefore, a DAXX-promoting phytoestrogen should be explored for prevention of tumor progression in advanced disease and relapse in the adjuvant setting. (PS2) transcripts were measured in cells-expressing or depleted for DAXX. Neither naringenin, resveratrol, or quercetin induced (PS2) expression to the same level as E2 (Supplementary Fig. 2). The modest increase in PS2 transcripts was not dependent on DAXX expression (Supplementary Fig. 2). These findings indicate that phytoestrogens, naringenin and resveratrol, are sufficient to increase DAXX protein in ER+ breast malignancy cells without stimulating total tumor cell proliferation or significantly activating classical ER signaling. ER or DAXX are required for inhibition of TIC survival by phytoestrogens To determine if naringenin or resveratrol inhibited survival of breast TICs through a DAXX-dependent manner, mammosphere forming efficiency (MFE) was performed on MCF-7 and T47D cells-expressing or depleted for DAXX. Expression of DAXX protein was detected by western blotting to confirm siRNA-mediated knockdown (Fig. ?(Fig.2a).2a). Results showed that E2, naringenin, or resveratrol increased DAXX protein expression compared to 3,5-Diiodothyropropionic acid the vehicle control (Fig. ?(Fig.2a).2a). Quercetin also increased DAXX protein but to a lesser degree than E2, naringenin, or resveratrol. The increased DAXX protein by E2, naringenin, resveratrol, or quercetin was almost completely abrogated by fulvestrant (Fig. ?(Fig.2a),2a), suggesting that this ER is required for DAXX protein expression. Results from MFE exhibited 3,5-Diiodothyropropionic acid that naringenin, resveratrol, or quercetin reduced survival of TICs similarly to E2 when compared to the vehicle control (Fig. ?(Fig.2b,2b, ?b,c).c). Further, DAXX or the ER was required for the reduction in TIC survival as DAXX depletion by siRNA or inhibiting ER function using fulvestrant, respectively, 3,5-Diiodothyropropionic acid rescued the decreased %MFE in response to E2, naringenin, or resveratrol (Fig. ?(Fig.2b,2b, ?b,c).c). In contrast, quercetin-mediated decrease in TIC survival was dependent on the ER but not on DAXX expression (Fig. ?(Fig.2b,2b, ?b,c).c). Together these findings indicate that phytoestrogens naringenin and resveratrol are sufficient to restrict TIC survival in an ER and DAXX-dependent manner. However, other phytoestrogens such as quercetin are potent inhibitors of TICs, but in a DAXX-independent manner. Open in a separate window Fig. 2 ER or DAXX are required for inhibition of TIC survival by phytoestrogens.a MCF-7 3,5-Diiodothyropropionic acid and T47D cells were transfected with a control (SCBi) or DAXX (DAXXi) siRNA for 48?h followed by treatment with a vehicle (ethanol), 5?nM E2, E2?+?100?nM fulvestrant (FULV), 100?nM naringenin (NG), 100?nM resveratrol (RES), or 100?nM quercetin (Q) alone or plus fulvestrant for 3 days. Western blotting was performed to detect DAXX and -Actin proteins. Images are representative of three impartial studies. b Cells were then plated onto low-attachment, six-well plates made up of methylcellulose, mammosphere forming medium at a density of 50,000 cells/well. Plates were incubated at 37?C for 7 days. Percent mammosphere forming efficiency was calculated based on the # of mammospheres counted/# of cells seeded??100. The bar graphs are the mean??s.d. of three impartial studies. Statistical significance between groups was analyzed using a One-way ANOVA. The asterisk denotes significance between E2, NG, RES, or Q and the ?E2 group under control siRNA (SCBi) conditions (and through a DAXX-dependent mechanism, ER+ MCF-7 and T47D cells-expressing or depleted for DAXX were treated with naringenin, resveratrol, or quercetin and NOTCH4 protein and NOTCH target gene transcripts were measured. As previously shown, E2 is sufficient to maintain high DAXX protein.