2016; 7:42777C42791. D, E) Downstream targets of RANK were analyzed by western blot upon stimulus with 1 g/ml RANKL for the indicated time points. -Actin was used as loading control. (F) Doubling time was quantified under standard conditions, and calculated using exponential growth equation with least squares regression fitting model (= 3). (G) Western blot of ER with -Actin as loading control. (H) Cell viability was measured after 5 days of culture in steroids-depleted medium +/C 10 nM -estradiol (= 3). (I) Western blot analysis of cell cycle-related proteins with -Actin as loading control. (J) Cell viability was measured 7 days after exposure A-1165442 to tamoxifen or fulvestrant, with medium replacement every SMAX1 48 h. (= 3). (K) Representative western blot of down-stream target of fulvestrant (ER) with -Actin as loading control (= 3). FiJi was used to obtain the best contrast for western blot band visualization, and background was removed for band densitometry analysis. Results are presented as the mean SEM. * < 0.05, ** < 0.01, *** A-1165442 A-1165442 < 0.001. Exposure to exogenous RANKL had no effect on luminal cells proliferation; however, RANK OE cells were less proliferative upon release from serum starvation in comparison with parental counterparts (Supplementary Figure 1E). We therefore quantified each cell lines doubling time, which was higher in RANK OE cells (Figure 1F). Since proliferation rate was negatively affected, we questioned if RANK OE impacts the expression of ER, a major regulator of proliferation in ER+ cells. We analyzed ER levels by western blot, and found ER to be up-regulated in RANK OE cell lines, although to a higher extent in MCF-7 cells (Figure 1G). However, upon estradiol deprivation RANK OE cells were significantly less sensitive to estradiol (Figure 1H). This may contribute to the decreased growth rate, and suggests that alternative pathways are involved in survival. To assess if RANK OE effects other proteins involved in cell cycle regulation, we synchronized cells in G0-G1 by serum starvation, followed by serum starvation-release with 10%FBS for 24 h (Supplementary Figure 1F). Comparison of MCF-7 and MCF-7OE cells shows a decrease in CDK2, p27 and p18 in RANK OE cells (Figure 1I). Moreover, serum starvation for 24 h had a very discrete effect in MCF-7OE cells. Comparison of T47D and T47DOE cells shows an increase in cyclinD1 and p21, and down-regulation of p27 and p18, in RANK OE cells. Again, serum starvation for 24 h had a very discrete effect in T47DOE cells, in opposite to T47D cells. This suggests the existence of compensatory mechanisms in RANK OE cells to sustain proliferation in stress conditions. Because RANK OE cells were characterized by increased expression of ER but decreased sensitivity to estradiol, we questioned if this would affect the response A-1165442 to HT, standard of care for ER+ breast cancers in all settings. Drug sensitivity assays demonstrate that RANK OE cells had decreased sensitivity to fulvestrant but not to tamoxifen (Figure 1J). Tamoxifen is a selective estrogen receptor modulator (SERM), an agonist that allows partial activation of ER. Fulvestrant is, however, is a selective estrogen receptor down-regulator (SERD), a pure antagonist which competitively binds to ER and, in contrast to tamoxifen, induces a rapid degradation and loss of the ER protein. Since fulvestrant induces ER degradation A-1165442 in a dose dependent manner [23] and RANK OE cells overexpress the receptor, we hypothesized that fulvestrant was less effective due to sustained ER expression upon treatment. We confirmed our hypothesis by measuring ER in fulvestrant-treated cells, which was maintained at higher levels in comparison to parental cells (Figure 1K). This reinforces that other pathways are involved in survival of RANK+ ER+ breast cancer cells. RANK overexpression in ER+HER2- breast cancer cell lines induces mesenchymal and staminal characteristics Next, and based on the reported effects of RANK OE in TNBC, we analyzed the expression of known epithelial and mesenchymal cell markers and observed an increase in Snail in both RANK OE cell lines (Figure 2A); whereas increased expression of N-cadherin, Vimentin and Slug was observed in MCF-7OE cells, but not in T47DOE. We hypothesize that this can be due to the fact that T47D cells exhibit a more mesenchymal phenotype in comparison with MCF-7, with basal increased levels.