Twist is a transcriptional repressor of E-cadherin gene expression in breast cancer

Abstract

Twist is a basic helix loop helix protein that plays a role both in human development and in cancer biogenesis. While characterizing the effects of Twist on breast epithelial cell transformation, we identified E-cadherin as a target gene that is down-regulated by Twist. In this study, we demonstrate that Twist can transcriptionally repress E-cadherin in breast cancer cells. Using transient promoter assays, we show that Twist can down-regulate E-cadherin promoter activity by up to two folds. This is further supported by immunoblot analyses which indicates that over-expression of Twist decreases E-cadherin protein levels in breast cancer cell lines. Subsequently, chromatin immunoprecipitation performed on MCF-7/Twist and Hs578 T (high level of endogenous Twist expression) confirmed Twist binding to the E-cadherin promoter. Finally, the functional relevance of this regulation was verified by quantitative real-time PCR and immunohistochemistry on a cohort of breast cancer samples.

Figure 1. Twist inversely correlates with E-cadherin expression in breast cancer cells

A, Quantitative real-time PCR of breast cancer cell lines. Relative expression is calculated with MCF-7 as baseline. An inverse correlation was observed between Twist and E-cadherin mRNA expression as demonstrated by quantitative real-time PCR. B, Immunoblots showing E-cadherin and Twist expression in a panel of normal breast and breast cancer cell lines, which include both invasive and non-invasive phenotypes. Normal cell lines are seen on the left with progressively more invasive cell lines on the right. Antibodies against Twist were made in-house while E-cadherin (BD Biosciences) and β-actin (Sigma-Aldrich) were commercially obtained.

Figure 2. Twist repression of E-cadherin promoter

A, Dose dependent repression of E-cadherin promoter. Determination of the degree of suppression of the full-length E-cadherin promoter activity in the presence of increasing amounts of Twist plasmid in transient co-transfection assays. The Y103X mutant Twist construct (Mut) was used to test for specificity of repression. B, Upper panel shows a schematic of the E-cadherin promoter with E boxes shown as hollow triangles. Deletion constructs are indicated at 5′ end with E1–E7. Twist represses E-cadherin transcriptionally. Each of the 6 E-cadherin promoter reporter constructs was repressed 1.2 to 2 fold by Twist. The largest significant repression was seen in construct E4, indicating the importance of the proximal region in E-cadherin regulation by Twist. Statistical significance (P<0.05) is indicated by asterisks. C, Full length Twist is essential for E-cadherin repression. Upper panel shows E-cadherin E4 construct with E-box mutants indicated by M1, M2, and M3. Twist repression was statistically significant only in M1 construct indicating its lack of function in mediating the repression. This indicates that E-box 2 and 3 may play an important role in the down-regulation of E-cadherin by Twist. D, Upper panel shows Twist deletion mutants. Mutations are indicated with amino acids changed to stop codons. Lower panel shows mutant Twist constructs were unable to repress E-cadherin promoter significantly indicating that regions other than the basic helix loop helix domain play a role in the repression of E-cadherin.

Figure 3. In vivo binding of Twist protein to the E-cadherin promoter sequence

ChIP was carried out following established protocols using MCF-7/Twist and Hs578 T cells and analyzed using E-cadherin promoter-specific primers by PCR. Primers amplified a 93 base pair fragment. Identical volumes from the final precipitate were used for the PCR reactions. M-molecular weight marker (bp), lane 1- total input immunoprecipitate, lane 2- acetylated histone H3 immunoprecipitate, lane 3- no chromatin immunoprecipitate, lane 4- no antibody immunoprecipitate and lane 5- Twist immunoprecipitate.

Figure 4. Relation between Twist and E-cadherin expression in patient breast cancer samples

A, mRNA levels of Twist and E-cadherin in breast tumors measured by quantitative real-time PCR. A total of 31 breast tumor (grade I=6, grade II=12, grade III=13) and four normal breast samples were analyzed. Loss of E-cadherin expression was concomitant with increased Twist expression in higher-grade tumors. B, A trend towards an inverse correlation was observed between Twist and E-cadherin expression (r=−0.27, p=0.14) in the breast cancer patient samples. C, Results of immunohistochemical analysis of Twist and E-cadherin expression in eighty-seven human breast cancer samples. The crosstab shows comparison of Twist nuclear staining and its intensity (classified as low vs. medium/high) against E-cadherin expression (absent/low/medium vs. high). D, Representative photomicrographs of human breast tumor samples from lobular and ductal samples that were analyzed for Twist and E-cadherin levels (left panel − 40×, middle and right panel −20×).