Snail2 is an essential mediator of Twist1-induced epithelial mesenchymal transition and metastasis

Abstract

To metastasize, carcinoma cells must attenuate cell-cell adhesion to disseminate into distant organs. A group of transcription factors, including Twist1, Snail1, Snail2, ZEB1, and ZEB2, have been shown to induce epithelial mesenchymal transition (EMT), thus promoting tumor dissemination. However, it is unknown whether these transcription factors function independently or coordinately to activate the EMT program. Here we report that direct induction of Snail2 is essential for Twist1 to induce EMT. Snail2 knockdown completely blocks the ability of Twist1 to suppress E-cadherin transcription. Twist1 binds to an evolutionarily conserved E-box on the proximate Snail2 promoter to induce its transcription. Snail2 induction is essential for Twist1-induced cell invasion and distant metastasis in mice. In human breast tumors, the expression of Twist1 and Snail2 is highly correlated. Together, our results show that Twist1 needs to induce Snail2 to suppress the epithelial branch of the EMT program and that Twist1 and Snail2 act together to promote EMT and tumor metastasis.

Figure 1. Twist1 indirectly suppresses E-cadherin transcription to promote EMT

A. Bright-field images of HMLE cells expressing Twist1-ER or a control vector before and after 12 days of 20 nM 4-hydroxytamoxifen (4-OHT) treatment. B. Lysates from HMLE cells expressing Twist1-ER or a control vector were collected before and after 18 days of treatment with 4-OHT, analyzed by SDS-PAGE, and probed for Twist1-ER protein, E-cadherin, β-catenin, γ-catenin, fibronectin, vimentin, N-cadherin, and α-tubulin. C. Real-time PCR analysis of E-cadherin mRNA expression in HMLE cells expressing Twist1-ER or a control vector treated with 4-OHT. D. Lysates from HMLE cells expressing Twist1-ER or a control vector were collected during 12 days of 4-OHT treatment, analyzed by SDS-PAGE, and probed for E-cadherin and α-tubulin. HMLE cells expressing Twist1 or a control vector are used as the negative or positive control.

Figure 2. Induction of the transcription factor Snail2 is required for Twist1-induced EMT

A. Real-time PCR analysis of Snail2 mRNA expression in HMLE cells expressing Twist1-ER or a control vector treated with 4-OHT and in HMLE-Twist1-ER cells expressing shSnail2-1, shSnail2-2 or shControl during 12 days of 4-OHT treatment. B. Lysates from HMLE cells expressing Twist1 or a control vector, from HMLE cells expressing Twist1-ER, and from HMLE-Twist1-ER cells expressing shSnail2-1, shSnail2-2 or shControl during 12 days of 4-OHT treatment were collected, analyzed by SDS-PAGE, and probed for Twist1 or Twist1-ER, Snail2, and α-tubulin. C. Quantification of the Snail2 protein level shown in the bottom panel of B. The relative Snail2 protein level was calculated by normalizing the signal of Snail2 to α-tubulin, and the values at individual timepoints were compared to the value at Day 0 for each cell line. D. Bright-field images of HMLE-Twist1-ER cells expressing shSnail2-1, shSnail2-2 or shControl before and after 12 days of 4-OHT treatment.

Figure 3. Induction of Snail2 is required for suppression of E-cadherin during Twist1-induced EMT

A. Real-time PCR analysis of E-cadherin mRNA expression in HMLE-Twist1-ER cells expressing shSnail2-1, shSnail2-2 or shControl during 12 days of 4-OHT treatment. B. Lysates from HMLE-Twist1-ER cells expressing shSnail2-1, shSnail2-2 or shControl were collected during 12 days of 4-OHT treatment, analyzed by SDS-PAGE, and probed for E- cadherin and α-tubulin. C. Cells from HMLE-Twist1-ER cells expressing shSnail2-1, shSnail2-2 or shControl were immunostained for E-cadherin and β-catenin (green) and nuclei (DAPI, blue) at Day 0, 7 and 18 after 4-OHT treatment.

Figure 4. Suppression of Snail2 attenuates expression of mesenchymal markers during Twist1-induced EMT

A. Real-time PCR analysis of fibronectin, N-cadherin, and vimentin mRNA expression in HMLE-Twist1-ER cells expressing shSnail2-1, shSnail2-2 or shControl during 18 days of 4-OHT treatment. The final folds of induction are indicated in each graph. B. Lysates from HMLE-Twist1-ER cells expressing shSnail2-1, shSnail2-2 or shControl were collected during 12 days of 4-OHT treatment, analyzed by SDS-PAGE, and probed for fibronectin, N-cadherin, vimentin, and α-tubulin.

Figure 5. Snail2 is an evolutionally conserved direct transcriptional target of Twist1 in amniotes

A. Right panels are ChIP-Sequencing result showing the enriched Twist1-binding peaks at the +5kb to -5kb regions of the human Snail2, E-cadherin and Snail1 promoters. The Y-axis represents the amplitude of the ChIP-Seq signals. Left panel is a close-up view of the enriched Twist1-binding E-box sequence at the -306bp from the transcription start site of the human Snail2 promoter. B. HMLE-Twist1-ER cells were treated with 4-OHT for 4 days. Chromatin was immunoprecipitated using corresponding antibodies and PCR was performed using one primer set for the Twist1-binding region on the Snail2 promoter and one control set for GAPDH. C. The 606bp Snail2 promoter or the Snail2 promoter with E-box mutation were transfected with or without Twist1 and its dimerization partner E47 and processed for luciferase promoter assay. D. Alignment of the conserved Twist1-binding E-box (bold) in Snail2 promoters. The number at the start of the sequence indicates the distance from transcription start sites.

Figure 6. Induction of Snail2 is required for the ability of Twist1 to promote invasion and metastasis

A. Right panels are brightfield images of HMLE-Twist1 cells expressing shSnail2-1 or shControl. The left panel shows that their cell lysates were analyzed by SDS-PAGE, and probed for Snail2, Twist1, Ras, and β-actin. B. HMLE-Twist1 cells expressing shSnail2-1 or shControl were plated on Transwell to assay migration and on Transwell with Matrigel to assay invasion. The percentage of cells that migrated through the insert was quantified. Error bars are standard error of the mean (SEM). C. Top two panels are representative images of lungs from mice injected with HMLER-Twist1 cells expressing shSnail2-1 or shControl. The bottom panel is immunostaining for E-cadherin (brown) in primary tumors from HMLER-Twist1 cells expressing shSnail2-1 or shControl. D. The top graph is quantification of total number of GFP positive colonies in individual lungs. The bottom graph is total primary tumor weight in individual mice carrying tumors of HMLER-Twist1 cells expressing shSnail2-1 or shControl. Error bars are SEM. N = 6 mice for the shControl group, and N = 8 mice for the shSnail2-1 group. Error bars are SEM.

Figure 7. Twist1 and Snail2 are frequently co-expressed in human breast tumors

A. Lysates from SUM1315 cells expressing shTwist1 or shControl were analyzed by SDS-PAGE and probed for Twist1, Snail2, and β-actin. B. All 22283 gene probesets are ranked based on their expression correlation coefficients with Twist1 in their human breast tumor datasets STOCKHOLM (n=159), EMC (n=286), and UPPSALA (n=236) and their distributions are shown in individual graphs. The rank of Snail2 is marked as a vertical line in each graph. C. A model describing how induction of Snail2 by Twist1 suppresses E-cadherin and promotes EMT and tumor metastasis.