A. Total proteins from three tumorigenic (MCF-7, T-47D, and Hs 578T) and two metastatic (MDA-MB-231 and MCF-7/Twist) breast cancer cell lines were immunoblotted for Twist and ER. Antibodies against Twist were made in-house, while ER (Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Sigma-Aldrich) were obtained commercially.
B. Histogram depicting relative expression of Twist and ER mRNA from cell lines analyzed by qRT-PCR. The primers used were 5′-GGACAAGCTGAGCAAGATTCAGA-3′ and 5′-TCTGGAGGACCTGGTAGAGGAA-3′ for Twist and 5′-GACAGGGAGCTGGTTCACAT-3′ and 5′-AGGATCTCTAGCCAGGCACA-3′ for ER. Values for Twist and ER were normalized to values in MCF-7. Experiments were repeated thrice in duplicates. Error bars depict S.D.
C. Schematic representation of ER promoter constructs showing the location of putative Twist binding E-boxes, denoted by short vertical lines. Areas spanned by chromatin immunoprecipitation (ChIP) amplicons are denoted by lines with E-box numbers. Promoter reporter assay results of transient Twist transfections in MCF-7 cells are displayed in the histogram on the right. Experiments were repeated five times in duplicates. Error bars depict S.D.
D. Schematic displaying Twist wild-type (wt) and mutant constructs. Stop codons, the DNA binding basic domain (B), and the helix-loop-helix (HLH) domain are indicated. Promoter activities of the constructs are displayed on the adjacent histogram. All luciferase activities were normalized to the activity of wild type Twist. Experiments were repeated twice in duplicates. Error bars depict S.E.M.
E–F. Chromatin immunoprecipitation using Twist antibody was carried out using MCF-7/Twist and MDA-MB-231 cells and analyzed using E-box specific primers by quantitative real-time PCR. Histograms depict the amplification from each primer set as a percentage of input chromatin compared to IgG negative control. A rabbit monoclonal antibody against histone H3 was used as positive control.

A–C. Histograms depicting cell cycle phases for MCF-7 and MCF-7/Twist cells grown in the absence of estrogen (charcoal stripped serum).
D–F. Histograms displaying cell cycle profiles of MCF-7 and MCF-7/Twist cells grown in estrogen-free media followed by tamoxifen treatment.
G–I. Histograms depicting results of cell cycle analysis of MCF-7 and MCF-7/Twist cells following Fulvestrant treatment.
Cell cycle stage values were calculated by the Dean-Jett-Fox model (Fox 1980). Experiments were independently repeated four times.

A–C. Histograms depicting increased cell death in Twist down-regulated MCF-7/Twist cells in the absence of estrogen (charcoal stripped serum) and presence of tamoxifen.
D–F. Histograms displaying increasing cell death in MDA-MB-231 shTwist cells in the absence of estrogen (charcoal stripped serum) and in the presence of tamoxifen.

A. Line chart showing growth of 1 × 10⁶ MCF-7 cells (without E2, solid triangles; with E2, hollow circles) and MCF-7/Twist cells (without E2, solid squares) orthotopically implanted into female SCID mice and allowed to grow for the time indicated. Tumors were measured weekly.
B. MCF-7/Twist tumors from mice (n=4) were excised and Twist and ER transcript levels determined by qRT-PCR. The graph depicts relative differences in Twist and ER transcript levels.
C, D. Growth of MCF-7 and MCF-7/Twist tumors over eight weeks treated with tamoxifen. tamoxifen pellet implantation is indicated by an arrow.
E, F. Representative false color coded MRI generated 3-D transverse slices of MCF-7 and MCF- 7/Twist xenografts in the mammary fat pad. Red and green represent the distributions of vascular volume (VV) and vascular permeability surface area product (PS), respectively. Gray-scale images represent the mouse body; while tumors are seen on top and indicated by “T”. Averaged values from all mice are indicated in the figures as well as displayed on the histograms at the right. Images depicted are a representative sample of five mice for MCF-7(+E2), six mice for MCF-7/Twist(−E2), and six mice for MCF-7/Twist (+E2).

A,B. Histograms depicting changes in Twist and ER expression after Twist and shTwist mediated up- and down-regulation in MCF-7 and MCF-7/Twist cells respectively. Transcript levels were estimated by qRT-PCR and are derived from three independent experiments in duplicates. Error bars depict S.D.
C. A panel displaying immunoblots of Twist up- and down-regulated cell lines scored for Twist and ER.
D. Histogram depicting changes in relative binding of ER to ERE luciferase plasmid in MCF-7, MCF-7/Twist, and shTwist mediated Twist down-regulated MCF-7/Twist cells. Experiments were repeated thrice in duplicates. Error bars depict S.D.
E,F. Histogram of qPCR results displaying the effect of (E) Twist down-regulation on ER downstream target genes in MCF-7/Twist cells; (F) Twist up-regulation on downstream ER target genes in MCF-7 cells.
G. Immunoblots of ER downstream targets that are dysregulated in MCF-7/Twist cells compared to parental MCF-7 cells. Actin was used as a loading control.
H. Basal ER promoter methylation levels of MCF-7 (low Twist, high ER), MCF-7/Twist and MDA-MB-231 (high Twist, low ER). Experiments were repeated twice in duplicates. Error bars depict S.D.
I. Histogram displaying changes in ER promoter methylation in cell lines after Twist up- and down-regulation. Experiments were repeated twice in duplicates. Error bars depict S.D.
J. Histogram showing increase in ER expression in MCF-7/Twist cells treated with 1μM demethylating agent 5-azacytidine (AZA) and 10 μM HDAC inhibitor valproic acid (VPA) and assayed by qRT-PCR. Experiments were repeated twice in duplicates. Error bars depict S.D.
K. Immunoblots of ER re-expression in MCF-7/Twist cells after treatment by AZA, VPA, and in combination.
L. Immunoblots of co-immunoprecipitation of MCF-7/Twist lysates. Twist antibodies were used for the co-immunoprecipitation and immunoblots were probed with HDAC1 and DNMT3B.
M. Histogram of chromatin immunoprecipitation results of MDA-MB-231 cells using HDAC1 and DNMT3B antibodies. E-boxes covered in the ER promoter are indicated on the horizontal axis. Results are displayed compared to negative control IgG and are calculated as a percentage of total input DNA. Bars depict S.D.

A. Histogram depicting Twist and ER transcript levels in various grades of breast cancer from 73 patient samples (Grade 1, n=16; grade 2, n=22; grade 3, n=35). Error bars display S.D.
B. Scatter plot of results of qPCR from samples of grades 1 and 2 breast cancer.
C. Scatter plot of qPCR results from grade 3 samples.

A proposed model depicting the normal, active state of transcription with unmethylated promoter DNA and acetylated chromatin leading to an ER+, hormone sensitive state. On binding to its target E-boxes, Twist recruits HDAC1 that deacetylates histone proteins leading to compaction of chromatin. Twist (with HDAC1) also interacts with DNMT3B at the E-boxes that causes de-novo methylation of the promoter area. Together with other, as yet unknown co-factors, Twist binding eventually leads to a repressive state of transcription ultimately culminating in the shutting down of ER transcription.