CEA interacts with and disrupts the TGF-β pathway.

<p>(A) Overexpression of CEA B3 domain prevents TGF-β-induced Smad3 phosphorylation. HCT116 cells were co-transfected with CEA wild type (FL) or various CEA deletion mutants. The cells were stimulated with TGF-β1 for 2 hours. The cell lysates were immunoblotted with the indicated antibodies. (B) CEA transcriptionally regulates TGF-β pathways. HCT116 cells were transfected with CEA. The mRNA levels of TGF-β targets were detected by Q-RT-PCR. The result shown is representative of three independent experiments. (C) Overexpression of CEA B3 domain inhibits Smad3 transcriptional activity. HCT116 or SW837 cells were co-transfected with Smad3 luciferase reporter plasmids, CEA (FL, N-A3, and B3), and Renilla luciferase reporter plasmids. The cells were stimulated with TGF-β1 for 2 hours. Cell lysates were collected and analyzed according to the manufacturer’s protocol. Results are the average of three independent experiments and are presented as mean ± SD. *<i>p</i> < 0.01, compared with Flag-Smad3/TGF-β1 treatment in Vector-Ctrl cells. Student’s t-test. NS: not significant. (D) CEA knockdown increases TGFBR1 protein stability. DLD1 cells were knocked down with control or shCEA. Cells were treated with 100ug/ml cycloheximide (CHX) for the indicated times. The density of TGFBR1 or TGFBR2 and the integrated optical density were measured. The turnover of TGFBR1 or TGFBR2 is indicated graphically. (E) Knock down CEA and/or TGF-β treatment suppresses CRC cell growth. Cell proliferation was assessed by colorimetric MTS assays. Results are the average of three independent experiments and are presented as mean ± SD. *<i>p</i> < 0.01, Student’s t-test. (F) TGF-β significantly suppresses cell migration in DLD1 CEA knockdown cells, while increasing cell migration in control DLD1-shCtrl cells. Transwell migration assays of DLD1-shCtrl or DLD1-shCEA cells were performed. Cells were treated with TGF-β1 (200pM) for 24 hours. Results are the average of three independent experiments and are presented as mean ± SD. *<i>p</i> < 0.05, **<i>p</i> < 0.01, Student’s t-test. (G) Proposed model of the role of CEA in the regulation of TGF-β pathway.</p

Enhanced expression of CEA correlates with loss of TGF-β signaling in early colon adenomas.

<p>(A) Marked increases in CEA mRNA expression levels in the highly mutated adenoma samples, MDA27ad-TA and MDA34ad-TVA. CEA mRNA expression levels were detected in 4 adenomas (MDA27ad-TA, MDA34ad-TVA, MDA2ad-TVA, and MDA3ad-SSA) and 4 matched normal mucosa samples (MDA27nor, MDA34nor, MDA2nor, and MDA3nor) by Q-RT-PCR. Results are the average of three independent experiments and are presented as mean ± SD. *<i>p</i> < 0.01, versus normal tissues, Student’s t-test. (B) Analysis of an Oncomine™ dataset demonstrates high CEA mRNA levels in colon and rectal adenomas compared to that in corresponding normal colorectal tissue. CEA and CEACAM6 mRNA expression is inversely associated with TGF-β target gene levels in normal and adenoma colorectal tissues. Transcriptomic profiles of 32 colorectal adenoma tumors and their 32 corresponding normal colorectal tissue were downloaded from Gene Expression Omnibus database (data set GSE8671). These gene expression profiles were then analyzed using Oncomine analysis tools (<a href="http://www.oncomine.org/" target="_blank">www.oncomine.org</a>). Data are displayed as a heat map using an Oncomine™ graphic platform and as a dot plot using a GraphPad Prism v5.0 program. *: <i>p</i> < 0.05, Student’s t-test. (C) Adenoma samples analysis of CEA, TGFBR1, TGFBR2, and β2SP reveals a negative correlation between CEA and TGF-β genes in colon adenomas. Sections from human clinical samples of normal (n = 10) and adenoma colon tissue (n = 26) were prepared and processed for immunohistochemical analysis, with further confirmation using 40 adenomas. Magnification × 20; insets magnification × 60. Scale bars, 10 mm. Quantification of the immunohistochemical staining is shown. Mean ± SD is shown *: <i>p</i> < 0.01, Student’s t-test.</p

CEA interacts with the TGF-β pathway.

<p>(A) The CEA B3 domain is required for the interaction of CEA with TGFBR1. HCT116 cells were co-transfected with the generated mutants. Cell lysates were immunoprecipitated with the CEA antibody and were immunoblotted with the indicated antibodies. (B) CEA interacts with the extracellular domain of TGFBR1 (amino acid 1–104). The generated TGFBR1 mutant plasmids were co-transfected into the cell lines and immunoprecipitation was carried out with CEA antibodies, followed by immunoblotting. (C) The CEA B3 domain interacts directly with TGFBR1. The CEA B3 domain GST fusion protein was produced bacterially. HA-TGFBR1-FL or HA-TGFBR1-1-104 deletion plasmids were transfected in 293T cells. Purified TGFBR1-FL (full length) or HA-TGFBR1- del-1-104 was incubated with GST-empty or GST-CEA-B3 proteins. The binding of TGFBR1 to CEA-B3 domain was detected by immunoblotting using anti-HA antibody. * designates non-specific bands. Purified GST fashion proteins were detected by Coomassie blue staining. Glutathione Resin GST Fusion Protein Purification Kit (GenScript, Cat. L00206) was used for purification of GST-CEA-B3 domain fusion protein.</p