BMP4 Signaling Is Able to Induce an Epithelial-Mesenchymal Transition-Like Phenotype in Barrett's Esophagus and Esophageal Adenocarcinoma through Induction of SNAIL2

Abstract

Background: Bone morphogenetic protein 4 (BMP4) signaling is involved in the development of Barrett's esophagus (BE), a precursor of esophageal adenocarcinoma (EAC). In various cancers, BMP4 has been found to induce epithelial-mesenchymal transition (EMT) but its function in the development of EAC is currently unclear.

Aim: To investigate the expression of BMP4 and several members of the BMP4 pathway in EAC. Additionally, to determine the effect of BMP4 signaling in a human Barrett's esophagus (BAR-T) and adenocarcinoma (OE33) cell line.

Methods: Expression of BMP4, its downstream target ID2 and members of the BMP4 pathway were determined by Q-RT-PCR, immunohistochemistry and Western blot analysis using biopsy samples from EAC patients. BAR-T and OE33 cells were incubated with BMP4 or the BMP4 antagonist, Noggin, and cell viability and migration assays were performed. In addition, expression of factors associated with EMT (SNAIL2, CDH1, CDH2 and Vimentin) was evaluated by Q-RT-PCR and Western blot analysis.

Results: Compared to squamous epithelium (SQ), BMP4 expression was significantly upregulated in EAC and BE. In addition, the expression of ID2 was significantly upregulated in EAC and BE compared to SQ. Western blot analysis confirmed our results, showing an upregulated expression of BMP4 and ID2 in both BE and EAC. In addition, more phosphorylation of SMAD1/5/8 was observed. BMP4 incubation inhibited cell viability, but induced cell migration in both BAR-T and OE33 cells. Upon BMP4 incubation, SNAIL2 expression was significantly upregulated in BAR-T and OE33 cells while CDH1 expression was significantly downregulated. These results were confirmed by Western blot analysis.

Conclusion: Our results indicate active BMP4 signaling in BE and EAC and suggest that this results in an invasive phenotype by inducing an EMT-like response through upregulation of SNAIL2 and subsequent downregulation of CDH1.

Fig 1. Expression of BMP4, BMP4 pathway associated molecules and the downstream target ID2.

a. Q-RT-PCR was used to determine mRNA expression of BMP4, its downstream target, ID2, BMP4 associated receptors and SMAD molecules in SQ, BE and EAC biopsy specimens. B2M and GAPDH were used for normalization. Data are relative to the mean ΔCt of SQ biopsies and are expressed as box plots, representing the mean with the minimum and maximum values. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. b. Western blot analysis in SQ, BE and EAC biopsy specimens showed BMP4, and ID2 expression and phosphorylation of SMAD1/5/8 in BE and EAC. Actin was used as loading control. Biopsy samples from 6 EAC patients were used. Representative pictures are shown. c. IHC showed nuclear and cytoplasmic expression of SMAD4 in 10 of 13 BE (arrowhead) and 11 out of 13 EAC tissue sections. EAC* represents a biopsy specimen with positive SMAD4 staining, EAC** represents a biopsy specimen with negative SMAD4 staining, stromal cells are SMAD4 positive and serve as internal control. Haematoxylin counterstain was used. Representative pictures are shown.

Fig 2. ID2 expression upon BMP4 or Noggin incubation.

a. mRNA expression of the downstream target ID2. B2M was used for normalization. Data are relative to the mean ΔCt of control cells and are expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001. b. Western blot analysis of BAR-T and OE33 cells incubated with BMP4 showed upregulated ID2 expression and more phosphorylation of SMAD1/5/8. Cells incubated with Noggin showed downregulated ID2 expression. Actin was used as loading control.

Fig 3. Migration assays of BAR-T and OE33 cells upon incubation with BMP4 or Noggin.

a. In vitro scratch assay of BAR-T and OE33 cells. The rate of migration across the scratched area was monitored for 24 h. Representative images showed that the scratch induced cells incubated with BMP4 to migrate compared to control cells or cells incubated with Noggin. Images are representative of three independent experiments. b. Boyden chamber assay of BAR-T cells incubated with BMP4 or Noggin. Data are relative to control cells not incubated with BMP4 or Noggin and expressed as mean ±SD. * p<0.05, ** p<0.01.

Fig 4. mRNA and protein expression of factors associated with EMT upon BMP4 or Noggin incubation.

a. Q-RT-PCR was performed to determine mRNA expression of SNAIL2 and its target genes, CDH1, CDH2 and Vimentin. B2M was used for normalization. Data are relative to the mean ΔCt of cells incubated with Noggin and are expressed as mean±SEM. *p<0.05. b. Western blot analysis of BAR-T and OE33 cells showed that SNAIL2 expression was upregulated and CDH1 expression was downregulated in cells incubated with BMP4 compared to cells incubated with Noggin. Actin was used as loading control. Pictures are representative of three independent experiments.