1. Volume 137, Issue 1, Pages 165-175 (July 2009)
A Colorectal Cancer Expression Profile That Includes Transforming Growth Factor β Inhibitor BAMBI Predicts Metastatic Potential 
Johannes Fritzmann, Markus Morkel, Daniel Besser, Jan Budczies, Frauke Kosel, Felix H. Brembeck, Ulrike Stein, Iduna Fichtner, Peter M. Schlag, Walter Birchmeier 
Gastroenterology 
Volume 137, Issue 1, Pages (July 2009)
DOI: /j.gastro
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2. Figure 1
Gene expression signature separates metastatic from nonmetastatic colorectal carcinomas. (A) Unsupervised hierarchical clustering of gene expression profiles. Clustering orders the samples according to greatest similarity of gene expression (dendrogram). Black boxes indicate closely related pairs of primary tumors and metastases from the same patient. (B) Mean expression levels of 115 signature genes that distinguish metastatic from nonmetastatic primary tumors. Hierarchical clustering dendrogram is shown on top: average gene expression levels in metastatic (mPT) and nonmetastatic (nPT) primary tumors (inside black box), lymph node (LN), liver (Hep), and lung (Pul) metastases and normal tissue (N). Selected genes that are involved in signal transduction, gene transcription, or metastasis are indicated. (C) Linear discriminant analysis of signature genes to classify tumors. The red line discriminates between metastatic (above) and nonmetastatic classes (below).
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Expression of selected signature genes in nonmetastatic and metastatic tumors. (A) CDC28 protein kinase regulatory subunit 2 (CKS2), (B) Midkine (MDK), (C) nuclear factor, interleukin 3 regulated (NFIL3), and (D) heat shock protein 90-kilodalton β (Grp94), member 1 (HSP90B1). Expression, as determined by microarray analysis for each sample (blue circles). Arbitrary units are given; 1 denotes the median expression in the complete data set. Red bars, mean expression for each subgroup.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
BAMBI expression differentiates between metastatic and nonmetastatic tumors and predicts metastasis formation. (A) Expression of BAMBI (as in Figure 2A). (B) Kaplan–Meier survival analysis of patients whose tumors were examined in the microarray analysis (n = 40). Patients were grouped in high BAMBI expressers (more than 1.2-fold median expression) and low BAMBI (less than 1.2-fold). (C) Box plot that displays BAMBI expression in metastatic (mPT) and nonmetastatic primary tumors (nPT) from an independent patient cohort (50 primary tumors) as determined by quantitative RT-PCR. All patients were free of distant metastasis at the time of sample collection, but, in the mPT group, metastases developed during follow-up. (D) Kaplan–Meier survival analysis of patients of this independent cohort, using high and low BAMBI expression, as determined by qRT-PCR. (E) In situ hybridization of BAMBI in normal colon mucosa (left panels) and nonmetastatic (middle panels) and metastatic primary tumors (right panels). Two independent examples are shown for each group. Scale bar, 100 μm.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
BAMBI expression is regulated by β-catenin/BCL9-2 signaling and counteracts TGF-β signaling in colon cancer cells. (A) Northern blot of BAMBI expression in SW480 cells following transient RNAi against β-Catenin, BCL9, and BCL9-2 (in triplicates). Nontargeting siRNAs were used as control. For siRNA efficacy see Figure S4. 18S and 28S rRNAs are used as loading control. (B) TGF-β signal transduction in Smad4-reconstituted SW480 cells: effect of BAMBI or BCL9-2 RNAi. Smad4_C4 and Smad4_D6 are independent SW480 clones that express wild-type SMAD4. (C) TGF-β signal transduction in HCT116 and LS174T cells: effect of BAMBI or BCL9-2 RNAi. Cells in A and B were first transfected transiently with (CAGA)12-MLP-Luc and with siRNAs as indicated and then stimulated with TGF-β1. Results are presented as the mean of duplicates.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Interaction of BAMBI with TGF-β signaling influences cell motility and morphology. (A) BAMBI expression in parental HCT116 (control) and HCT116 cells that were stably transfected with BAMBI (clones BAMBI_A3 and BAMBI_C1) as analyzed by qRT-PCR. (B) TGF-β signaling in these BAMBI-expressing clones (HCT116-BAMBI_A3 and BAMBI_C1) (see Figure 4). (C) Regulation of the TGF-β target gene PMEPA1 in control and the BAMBI-expressing clone BAMBI_A3 after treatment with TGF-β1 or SB (SB). Levels of PMEPA1 were determined by qRT-PCR. (D) Cell migration of the BAMBI_A3 and BAMBI_C1 clones, relative to parental HCT116 cells. Number of migrating cells in transwell assays was determined in triplicates, fold induction (±SEM) compared with controls. (E) Cell morphology and actin cytoskeleton immunofluorescence of HCT116 cells following transfection of BAMBI or BCL9-2 siRNAs or treatment with TGF-β1 or SB431542, as indicated. Upper panels, phase contrast microscopy; lower panels, phalloidin immunofluorescence (red) and nuclear DAPI staining (blue).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
BAMBI expression promotes experimental metastasis. (A) Expression of BAMBI protein in stably transfected LS174T cell clones (BAMBI_C3, BAMBI_C4, BAMBI_C6). (B) Quantification of liver metastases following injection of BAMBI expressing clones (2 × 106 cells) into the spleen of nude mice. Number of metastases in the liver was determined after 4 weeks (mean ± SEM; n = 4 per group). (C) BAMBI mRNA and protein expression in primary tumors and liver metastases of xenografts as analyzed by in situ hybridization (upper panels) and immunohistochemistry (lower panels). (D) Quantification of lymph node metastases following intravenous injection of BAMBI expressing LS174T and control cells (1 × 106 cells). Shown is the metastasis index as fold increase over control, n = 8; error bars indicate SEM. (E) Group of nude mice from experiment in D (using BAMBI_C4 cells), showing lymph node metastases, visible at typical sites (left panel, black arrows). Examples of in situ lymph node metastases (lower right panel) and H&E staining (upper right panel) are shown. Scale bars, C, 200 μm; E, 1 mm.
Gastroenterology  , DOI: ( /j.gastro )
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