Volume 137, Issue 2, Pages 639-648.e9 (August 2009) ITF-2 Is Disrupted via Allelic Loss of Chromosome 18q21, and ITF-2B Expression Is Lost at the Adenoma-Carcinoma Transition  Andreas Herbst, Guido T. Bommer, Lydia Kriegl, Andreas Jung, Andrea Behrens, Endy Csanadi, Markus Gerhard, Christian Bolz, Rainer Riesenberg, Wolfgang Zimmermann, Wolfgang Dietmaier, Isabella Wolf, Thomas Brabletz, Burkhard Göke, Frank T. Kolligs  Gastroenterology  Volume 137, Issue 2, Pages 639-648.e9 (August 2009) DOI: 10.1053/j.gastro.2009.04.049 Copyright © 2009 Terms and Conditions

Figure 1 ITF-2B is overexpressed in adenomas and frequently down-regulated in colorectal carcinomas. (A) ISH of ITF-2B expression in the small intestine of APCmin/− mice. Low magnification shows staining of all tumors in the intestine. (B) ITF-2B ISH from adjacent sections of an intestinal APCmin/− adenoma. (C) Immunohistochemistry of ITF-2B expression in normal human colon and (D) an adenoma from a patient with familial adenomatous polyposis coli (FAP). The arrow indicates lymphocytes. (E) The expression of ITF-2B mRNA was measured in matched pairs of microdissected colorectal cancers and adjacent normal colorectal epithelium by real-time RT-PCR. Expression of ITF-2B mRNA in tumor tissue was normalized to its expression in normal colorectal epithelium of the same patient. Deregulation of β-catenin/Tcf-4 signaling was determined by nuclear staining of β-catenin. Bars represent mean ± SD of an experiment performed in triplicate. Asterisks depict samples with a relative expression level of .5 and below. (F) The expression of ITF-2B in human colon cancer cell lines was determined by immunoblotting. β-actin was used as a loading control. The presence of mutations in the APC or β-catenin genes is indicated by + or − signs. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Figure 2 Re-expression of ITF-2B in colon cancer cells attenuates proliferation. (A and B) Colorectal cancer cell lines were transfected either with ITF-2B or a control vector and selected for neomycin resistance for 4 weeks. Relative colony formation after ITF-2B expression was compared with control plates. Results are expressed as mean ± SD of an experiment performed in triplicate. (C) DLD1 cells were stably transfected with a doxycycline (Dox)-inducible expression construct encoding the transcription factor ITF-2B (clones #B1/#B2) or an empty control vector (clones #C1/#C2). The conditional cell lines were treated with Dox for 24 hours. Expression of ITF-2B was detected using an HA antibody. β-actin was used as a loading control. (D and E) DLD1/ITF2B clones were plated in semisolid agar medium and treated with Dox for 4 weeks. Results are expressed as mean ± SD of an experiment performed in triplicate. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Figure 3 ITF-2B induces G0/G1 cell cycle arrest and sensitizes cells to TRAIL-induced apoptosis. (A) DLD1/ITF2B and control cells were synchronized, treated with doxycycline (Dox), and analyzed for cell cycle distribution by flow cytometry. Histograms of representative experiments are shown. (B) Quantitative analysis of cells in the subG1-, G0/G1-, S-, and G2/M-fractions. Black bars correspond to untreated cells; grey bars represent cells treated with Dox. Results are expressed as mean ± SD of a representative experiment performed in duplicate. (C) DLD1/ITF2B cells were treated with Dox in combination with 0, 1, or 5 ng TRAIL. The sub-G1 fractions were determined. Mean values ± SD of an experiment performed in triplicate are shown. Student t test was used for the statistical analysis. (D) The immunoblot of cell lysates of the experiment shown in C demonstrates cleavage of caspase 8. The amounts of caspase 8 cleavage products were determined densitometrically using the Software ImageJ (National Institutes of Health, Bethesda, MD) and normalized to β-actin. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Figure 4 ITF-2B blocks cell cycle progression by induction of p21Cip1. (A) DLD1/ITF2B cells (clones #B1 and #B2) were treated with Dox. Expression of the proteins ITF-2B, p15INK4B, p16INK4A, p21Cip1, p27Kip1, and β-actin was analyzed by immunoblotting. (B) DLD1/ITF2B cells were treated with Dox, and expression of ITF-2B, p21Cip1, and β-actin was analyzed at the indicated time points by immunoblotting. (C) Immunoblot analysis of DLD1/Cip1 cells, which express p21Cip1 upon withdrawal of tetracycline (Tet). (D) The effect of p21Cip1 expression on cell cycle distribution was determined in DLD1/Cip1 cells by flow cytometry. The graph depicts the changes in the ratio of cells in the G0G1/S phase of the cell cycle. The means ± SD of 2 independent experiments are shown. (E) DLD1/ITF2B cells were incubated with a β-galactosidase siRNA or siRNA targeting CDKN1A mRNA and treated with Dox. The graph depicts the changes in the ratio of cells in the G0G1/S phase. The means ± SD of a representative experiment performed in duplicate are shown. (F) Immunoblot of cell lysates of the experiment shown in panel E demonstrates silencing of p21Cip1 by the siRNA directed against CDKN1A mRNA. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Figure 5 ITF-2B regulates CDKN1A transcription via E-boxes. (A) The diagram shows part of the CDKN1A gene including exon 1 and exon 2, E-boxes (grey dots), and p53-binding sites (white dots). The position and size of the CDKN1A promoter fragments (L, K, DA) are indicated. The diagram is drawn to scale. (B) HCT116 p53 WT and HCT116 p53 ko cells were transfected with the indicated constructs, and reporter gene activity was determined. (C) HCT116 cells were transfected with the CDKN1A reporter gene constructs K, K/DA, and mutated forms of K/DA. Bars in C and D represent the means ± SD of experiments performed in duplicate. (D) Binding of ITF-2B to sequences within intron 1 of the CDKN1A gene was confirmed by ChIP. DLD1/ITF2B cells (clone #B1) were stimulated with doxycycline for 16 hours, and chromatin was immunoprecipitated using the indicated antibodies. Immunoprecipitated chromatin was analyzed using a pair of primers covering E-boxes 9 to 12 (Cip1 #A), a Cip1 control primer pair (Cip1 ctrl), and a primer pair for GAPDH. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Figure 6 Loss of ITF-2B expression occurs at the adenoma to carcinoma transition. (A) ITF-2B protein is expressed in adenomas and lost in colorectal carcinomas. Adjacent sections of a representative adenoma (panels a–c) and colorectal carcinoma (panels d–f) were stained with antibodies targeting β-catenin, ITF-2B, and p21Cip1 using immunohistochemistry. (B) Statistical analysis of ITF-2B and p21Cip1 protein expression in adenomas and colorectal carcinomas. Fifteen adenomas and 67 colorectal carcinomas (International Union Against Cancer [UICC] stages I–IV) were stained with antibodies recognizing ITF-2B and p21Cip1. Tumor samples were sorted according to their stage (their order is identical in the upper and lower panels), and protein expression for each sample was scored. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Figure 7 LOH is frequently observed at the ITF2 locus. Five chromosomal markers located in the chromosomal region 18q were analyzed for LOH by PCR. The marker D18S35 is located within the ITF2 gene. Black fields denote LOH. The ITF-2B protein expression was determined by immunohistochemistry, and the score for each case is indicated. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Figure 8 Histone deacetylation contributes to down-regulation of ITF-2B in colorectal cancer. (A) The relative ITF-2B mRNA expression in the cell lines HEK293, Colo320, DLD1, and SW480 was measured using real-time RT-PCR and normalized to the ITF-2B mRNA expression in Colo320 cells. (B) The acetylation status of H3 histones within the ITF2 promoter correlates with the ITF2 mRNA expression. HEK293, Colo320, DLD1, and SW480 cells were subjected to chromatin immunoprecipitation using antibodies against Gal4 (control) and acetyl-histone H3. Immunoprecipitated chromatin was analyzed using primer pairs specific for the ITF2 promoter and the Cyclin D1 promoter. (C) Treatment of DLD1 and SW480 cells with the histone deacetylase inhibitor LBH589 restores ITF-2B mRNA expression. The relative ITF-2B mRNA expression was measured by real-time RT-PCR. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Supplementary Figure 1 ITF-2B mRNA is frequently down regulated in colon cancer cell lines and re-expression of ITF-2B interferes with cell viability. (A) ITF-2B mRNA expression is lost in the majority of colorectal carcinoma cell lines. The normalized expression of ITF-2B mRNA was measured by real-time RT-PCR and normalized to the ITF-2B mRNA expression in normal colon tissue. (B) Expression of ITF-2B interferes with cell growth. DLD1/ITF2B (clones #B1 and #B2) and control cells (clones #C1 and #C2) were treated with doxycycline (+Dox) and the proliferation index was measured after 96 hours using the MTT assay. The bars represent mean ± S.D. of a representative experiment performed in duplicates. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Supplementary Figure 2 ITF-2B regulates CDKN1A transcription via specific E-boxes. (A) The diagram shows part of the CDKN1A gene including exon 1 and exon 2, E-boxes (grey dots) and p53 binding sites (white dots). The position and size of the CDKN1A promoter fragments are indicated. The diagram is drawn to scale. (B) DLD1 and HCT116 cells were transfected with the CDKN1A reporter genes K, M, and L and reporter gene activity was determined. Bars represent mean ± S.D. of a representative experiment performed in duplicates. (C) HCT116 cells were transfected with the CDKN1A reporter gene constructs K, K/FL, K/A, K/B, K/C, K/D, and K/E, and fold induction was determined. Bars represent mean ± S.D. of an experiment performed in duplicates. (D) HCT116 cells were transfected with the CDKN1A reporter gene constructs K, K/FL, K/DA, K/DB, K/DC, K/DD, and K/DE, and fold induction was determined. Bars represent mean ± S.D. of an experiment performed in duplicates. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Supplementary Figure 3 Analysis of CpG island methylation with the ITF-2 promoter revealed no significant changes. DLD1 cells were treated with 5-Aza-2′-deoxycytidine (AzaC) for 48 hours and the methylation status of a putative CpG island overlapping with exon 1 of the ITF2 gene was analyzed. The methylation status of normal (N) and tumor (T) tissue of three colorectal cancers (1-3) with proven LOH on 18q and loss of ITF-2B expression was determined. Black dots represent methylated CpG dinucleotides; white dots stand for unmethylated CpG dinucleotides. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Supplementary Figure 4 Loss of p21Cip1 expression at the adenoma to carcinoma transition does not correlate with p53 expression. (A) Immunohistochemical staining of sections of normal and colorectal carcinoma tissue with a p53 antibody. (B) Statistical analysis of p53 and p21Cip1 protein expression in adenomas and colorectal carcinomas. 15 adenomas and 67 colorectal carcinomas (UICC stages I-IV) were stained with antibodies recognizing p53 and p21Cip1. Tumor samples were sorted according to their stage (their order is identical in the upper and lower panels) and protein expression for each sample was scored. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions

Supplementary Figure 5 ITF-2B, DCC, and Smad4 protein expression correlates in colorectal carcinomas. (A) Immunohistochemical staining of sections of colorectal cancers with antibodes recognizing the proteins DCC and Smad4, respectively. (B) The table contains the expression scores for ITF-2B, DCC, and Smad4 as determined immunohistochemistry. The correlation between ITF-2B and DCC, as well as ITF-2B and Smad4 was determined. Gastroenterology 2009 137, 639-648.e9DOI: (10.1053/j.gastro.2009.04.049) Copyright © 2009 Terms and Conditions