Genes specifically perturbed by YC-1 in long-term estrogen deprivation of MC7 cells and their link to the response to endocrine therapy. (A) Genes whose expression change differentiates the effect of YC-1 between long-term estrogen deprivation of MCF7 (MCF7-LTED) cells and MCF7 cells. The bottom heatmap shows the normalized expression differences for the probes and genes that passed the defined thresholds. DMSO, Dimethyl sulfoxide. (B) Logarithmic fold changes between the responder and nonresponder breast tumors for the genes (Gene Expression Omnibus data set [GSE:32222]) shown in (A) determined by chromatin immunoprecipitation (ChIP) assay. (C) ChIP assay results for estrogen receptor α (ERα) and immunoglobulin G (IgG) at two sites in the VAV3 locus, both for MCF7 and MCF7-LTED cells with or without exposure to YC-1 (significant differences are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001). The bottom graph shows the genomic locus with the linkage disequilibrium structure in Japanese individuals found in HapMap and the relative position of the variant rs10494071 (presented below). (D) Detailed analysis of the Gene Expression Omnibus [GSE:32222] data set for the two sites depicted above. Left panels show the normalized average intensity of ERα binding ±500 bp around the sites in different sample sets as depicted in the insets. Middle panels show relative ERα binding in the above sites across 23 breast cancer samples. Right panels are graphs showing the number of cases with or without an ERα binding event (peak) in nonresponders and responders. Top right panels show that 44% of the nonresponders had ERα binding, whereas only two (22%) of nine responders had binding. Bottom right panels show that 78% of the nonresponders had ERα binding, whereas only one (11%) of nine responders had binding. (E) Short hairpin RNA (shRNA)–mediated depletion of ERα led to a decrease in VAV3 levels in MCF7-LTED cells, but not in MCF7 cells.