TAK1 negatively regultes ESCC migration and invasion

(A) Increased expression of TAK1 in ECA-109 cells transfected with a plasmid expressing Map3k7.

(B) Increased expression of TAK1 inhibits the morphological changes to form spindle-shaped mesenchymal cells induced by EGF (100 ng/ml) in ECA-109. Scale bar = 100 µm.

(C) Increased expression of TAK1 inhibits cell migration and invasion in ECA-109 cells. Cell migration and invasion were analyzed by transwell assay. n = 4 biologically independent replicates.

(D) Wound healing assay showing cell migration was attenuated by TAK1. n = 5 biologically independent replicates.

(E) TAK1 decreased mesenchymal marker gene expression, while increased the expression of epithelial markers. ECA-109 cells were transfected with the plasmid carrying Map3k7. 24 h post-transfection, protein samples were prepared and subjected to western blot. Actin was used as a loading control.

(F) Knockdown of TAK1 increased the expression of F-Actin. ECA-109 cells were transfected with Map3k7 siRNA. Seventy-two h post-transfection, cells were subjected to immunofluorescence analysis using an anti-F-Actin antibody (red). Hoechst was used to stain the nucleus (blue). Scale bar = 10 µm.

(G) TAK1 knockdown induces spindle-shaped mesenchymal cell morphology in ECA-109 cells. Scale bar = 100 µm.

(H) Reduced expression of TAK1 promotes cell migration and invasion. ECA-109 cells were transfected with Map3k7 siRNA. 72 h post-transfection, cell migration and invasion were analyzed by transwell assay. n = 5 biologically independent replicates.

(I) Knockdown of TAK1 increases mesenchymal protein marker expression, and decreases epithelial protein marker expression.

Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p < 0.05, **p < 0.01, and ***p < 0.001.

TAK1 phosphorylates PLCE1 at serine 1060

(A) Tandem mass spectrometry showing serine 1060 (S1060) in PLCE1 was phosphorylated by TAK1. ECA-109 cells were transfected with a plasmid expressing Map3k7. Twenty-four h-post transfection, cells were harvested and subjected to co-immunoprecipitation. The resulting immunocomplex was analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).

(B) TAK1 fails to phosphorylate PLCE1S1060A. ECA-109 cells were co-transfected with the plasmids carrying wildtype (WT) Plce1, mutated Plce1 (PLCE1S1060A), or Map3k7 as indicated. Twenty-four h-post transfection, cells were collected for western blot analysis

(C-E) Inhibition of TAK1 reduces PLCE1 phosphorylation at S1060. ECA-109 cells were co-transfected with the plasmids expressing Plce1 or Map3k7. 6 h post-transfection, TAK1 inhibitor (5Z)-7-Oxozeaenol (Oxo; 10 µM) (C), or 10 µM Takinib (D), or 10 µM NG25 (E) was added in culture medium, and cells were cultured for additional 18 h. Cells were then subjected to western blot analysis. Actin was used as a loading control

(F-G) Immunohistochemical analysis of TAK1 (F) and p-PLCE1 (G) expression in normal and esophageal squamous tumor tissues. n = 4 biologically independent replicates. Scale bar = 20 µm.

(H) Correlation between p-PLCE1 and TAK1 based on immunohistochemical data as shown in (F-G). 10 views for each sample were randomly chosen for Pearson correlation test.

(I) TAK1 and p-PLCE1 protein levels in clinical samples. Protein levels were analyzed by western blot, and Actin was used as a loading control. n = 4 biologically independent replicates. N: normal tissue; T: tumor tissue.

PLCE1 positively regulates ESCC migration and invasion

(A) Increased expression of PLCE1 in ECA-109 cells transfected with a plasmid expressing Plce1.

(B-C) Increased expression of PLCE1 enhances cell migration and invasion. ECA-109 cells were transfected with the plasmid carrying Plce1. Twenty-four h-post transfection, cells were subjected to transwell (B) or wound healing (C) assay. n = 5 biologically independent replicates.

(D) Increased expression of PLCE1 in ECA-109 cells induces mesenchymal protein marker expression, while reduces epithelial protein marker expression.

(E-F) Knockdown of PLCE1. ECA-109 cells were transfected with siRNAs targeting Plce1. Seventy-two h-post transfection, cells were harvested for analyzing PLCE1 expression by qRT-PCR (E) and western blot (F).

(G-H) Reduced expression of PLCE1 inhibits cell migration and invasion. ECA-109 cells were transfected with Plce1 siRNA-2. Forty-eight h-post transfection, cells were subjected to transwell (G) or wound healing (H) assay. n = 3-5 biologically independent replicates.

(I) Knockdown of PLCE1 promotes epithelial protein marker expression, while represses mesenchymal protein marker expression. ECA-109 cells were transfected with Plce1 siRNA-2. Seventy-two h-post transfection, cells were harvested and subjected to western blot analysis. Protein level was detected by western blot, and Actin was used as a loading control. Gene expression was analyzed by qRT-PCR and Gapdh was used as a house-keeping gene. Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p < 0.05, **p < 0.01, and ***p < 0.001.

TAK1 inhibits PLCE1 enzyme activity

(A-B) Effects of TAK1 on PLCE1 (A) and PLCE1 S1060A (B) enzyme activity. ECA-109 cells were co-transfected with the plasmids expressing Plce1-Myc or Map3k7. Twenty-four h post-transfection, cells were subjected to pull down assay by using the beads with anti-Myc antibody. PLCE1 enzyme activity was assayed by Phospholipases C (PLC) Activity Assay Kit. n = 3 biologically independent replicates.

(C-E) TAK1 abolishes PLCE1-induced IP3 and DAG in ECA-109 (C), KYSE-150 (D), and TE-1 cells (E). Cells were transfected with the plasmids bearing Plce1 or Map3k7 as indicated. Twenty-four h post-transfection, cells were harvested for measuring IP3 and DAG. n = 3 biologically independent replicates.

(F) TAK1 attenuates PLCE1-induced intracellular Ca2+ ([Ca2+]). ECA-109 cells were transfected with the plasmids bearing Plce1 or Map3k7 as indicated. [Ca2+] was labeled with Fluo-4 AM, which was then detected by a fluorescent microscope. Scale bar = 20 µm.

(G) Quantified fluorescence intensity of [Ca2+] in ECA-109 cells. n = 3 biologically independent replicates.

(H) Fluorescence intensity of Fluo-4 in ECA-109, KYSE-150, and TE-1 cells was examined with a fluorospectrophotometer. n = 3 biologically independent replicates.

(I) Flow cytometry analysis of [Ca2+]. Cell treatments were described in (F).

Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test (A-B) or two-tailed one-way ANOVA test (C-E, G-I). *p < 0.05, **p < 0.01, and ***p < 0.001.

TAK1 inhibits PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin

(A) IP3R blocking inhibits PLCE1 induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 h and then treated with 2-APB (10 µM) for additional 18 h.

(B) [Ca2+] blocking represses signal transduction in the axis of PKC/GSK-3β/β-Catenin induced by PLCE1. ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 h and then treated with BAPTA-AM (10 µM) for additional 18 h.

(C) PKC inhibition blocks PLCE1 stimulated signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmid expressing Plce1. 6 h post-transfection, cells were treated with 100 nM of Midostaurin for additional 18 h.

(D) PKC inhibition represses PLCE1-induced nuclear translocation of β-Catenin in ECA-109 cells. Cells were transfected with the plasmid expressing PLCE1. Six h post-transfection, 2-APB (10 µM), BAPTA-AM (10 µM), or Midostaurin (100 nM) was added in culture medium, and cells were cultured for additional 18 h. Scale bar = 10 µm. Immunofluorescence was used to examine subcellular distribution of β-Catenin.

(E) TAK1 counteracts PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmids expressing Plce1 or Map3k7 as indicated for 24 h.

(F) TAK1 reduces PLCE1-induced nuclear distribution of β-Catenin in ECA-109 cells. Cells were transfected with the plasmids expressing Plce1 or Map3k7 as indicated. Scale bar = 10 µm.

(G) Dominant negative TAK1 (K63W) fails to block signal transduction in the axis of PKC/GSK-3β/β-Catenin/MMP2 induced by PLCE1. ECA-109 cells were transfected with the plasmids expressing Plce1 or mutated Map3k7 (TAK1 K63W) for 24 h.

(H) TAK1 has no effect on PLCE1 S1060A induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmids expressing PLCE1 S1060A or TAK1 for 24 h. Protein levels were analyzed by western blot, and Actin was used as a loading control. Representative blots were shown.

Inhibition of TAK1 by Takinib promotes ESCC metastasis in nude mice

Each mouse was intravenously injected with 1 x 106 ECA109 cells diluted in 100 µl PBS. Mice were treated with Takinib at the dosage of 50 mg/kg/day for 15 days, mice in control group were received vehicle (corn oil). Eight weeks later, mice were sacrificed, and the lungs and livers from each group were collected and photographed.

(A) Typical images of specimens.

(B) Hematoxylin and eosin staining of metastatic nodules in lungs.

(C) The number of nodules in lungs.

(D) Takinib treatment induces signal transduction in the axis of PKC/GSK-3β/β-Catenin. Protein levels were analyzed by western blot, and Actin was used as a loading control. n = 3 biologically independent replicates.

(E) Quantitative analysis of the western blot data shown in (D).

Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. *p < 0.05, **p < 0.01, and ***p < 0.001.

PLCE1 knockdown inhibits ESCC metastasis in nude mice

ECA-109 cells were transduced with lentivirus bearing PLCE1 shRNA (LV-shPLCE1) or NC shRNA (LV-shPLCE1 NC). Each mouse was intravenously injected with the LV transduced cells (1 x 106 cells/mouse). Eight weeks later, mice were sacrificed, and the lungs and livers from each group were collected and photographed.

(A) Typical images of lung specimens.

(B) Hematoxylin and eosin staining of metastatic nodules in lungs.

(C) The number of nodules in lungs.

(D) PLCE1 knockdown represses signal transduction in the axis of PKC/GSK-3β/β-Catenin. Protein levels were analyzed by western blot, and Actin was used as a loading control. n = 3 biologically independent replicates.

(E) Quantitative analysis of the western blot data shown in (D).

Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. **p < 0.01, and ***p < 0.001.