Volume 142, Issue 4, Pages 875-885.e3 (April 2012) Cdx2 Controls Expression of the Protocadherin Mucdhl, an Inhibitor of Growth and β- Catenin Activity in Colon Cancer Cells  Isabelle Hinkel, Isabelle Duluc, Elisabeth Martin, Dominique Guenot, Jean–Noel Freund, Isabelle Gross  Gastroenterology  Volume 142, Issue 4, Pages 875-885.e3 (April 2012) DOI: 10.1053/j.gastro.2011.12.037 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Mucdhl expression depends on Cdx2. (A) Schematic representation of human Mucdhl L and M (CAD, cadherin motifs; IC, intracellular domain; MUC, mucin-like tandem repeats; S, signal peptide; TM, transmembrane domain). (B) A monolayer of Caco-2/TC7 cells was wounded, and, 48 hours later, cells located at the wound edge (M) or in the confluent monolayer (C) were separately collected for RNA extraction and RT-PCR with TBP as internal control. (C) HT29 were cultivated in vitro (V) or subcutaneously grafted (G) before RNA extraction and RT-PCR. (D) Caco-2/TC7 cells were grown for the indicated time before Western blot analysis. The multiple bands detected by the anti-Mucdhl antibody reflect as reported the presence of several isoforms and glycosylation.19–21 (E) Cdx2 was overexpressed (+) by transfection before RNA extraction and RT-qPCR. Results are presented as fold of induction for each cell line, with the level of Mucdhl expression (±standard deviation, normalized with TBP expression) in cells without Cdx2 overexpression set at 1. (F) SW480 cells were transfected with control (−) or Cdx2-specific small interfering RNA before RNA extraction and RT-qPCR. Results are presented as percentage of expression with the level of Cdx2 or Mucdhl expression (±standard deviation, normalized with TBP) in the control sample set at 100%. Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Cdx2 regulates the transcription of Mucdhl. (A) Chromatin of HCT116 cells expressing Cdx2 (+) or not (−) was subjected to immunoprecipitation (ChIP) with an anti-Cdx2 antibody or control IgG before PCR detection of a Mucdhl or a sucrase-isomaltase (SI, positive control) promoter fragment. PCR was also performed with 1% of the bulk DNA (input) to ensure equivalence of the samples. (B) As in panel A with Caco2/TC7 grown for 2 or 19 days. (C) Schematic representation of the different Mucdhl reporter plasmids tested with the positions of the potential Cdx2-binding sites (intact, shaded ovals; mutated, X). (D) HCT116 were transfected with a Cdx2-expressing plasmid (+) or the corresponding empty plasmid (−), the indicated pMucdhl-Luc reporter plasmid, as well as a promoter-less renilla reporter plasmid (internal control) before lysis and luminescence measurement. Relative promoter activity (±standard deviation, normalized with renilla values) of each of the pMucdhl-Luc reporter in the absence of Cdx2 was set at 1. (E) As in panel D, using the indicated reporter plasmids. * P < .01 and ** P < .001. Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Expression of Mucdhl in wild-type and Cdx2+/− intestines. Cdx2 and Mucdhl antibodies were used on paraffin-embedded sections. (A) Staged mouse embryos. (B) Adult human intestines. (C) Matching areas of normal intestinal epithelium and foregut-type heterotopia of a Cdx2+/− mouse. Original magnification, ×200, except in panel B with ×100 (left panel) and ×200 (right panel). Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Mucdhl expression is reduced in colon tumors. (A) H&E staining and immunohistochemistry were performed on tumor tissue from the distal colon of azoxymethane (AOM)-treated mice or APCΔ14/+ mice. (B) H&E and immunohistochemistry were performed on normal colon and colon tumors from 2 patients (original magnification, ×200). (C) RT-qPCR were performed on RNA from matched normal (circles) or tumor (squares) colon tissues. Relative Mucdhl expression (±standard error mean, normalized with TBP) for each normal colon was set at 1. Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Mucdhl can reduce colony formation of colon cancer cells. (A) Western blot showing expression of Mucdhl induced by doxycycline in representative HCT116 clones. Multiple bands reflect glycosylation of Mucdhl. (B) Control or Mucdhl-expressing clones were seeded at low density and cultivated in absence (−) or presence of doxycycline (+) in the medium. Results of a representative experiment performed in triplicates are shown. Number of colonies (±standard deviation) counted in the absence of doxycycline for each clone was set at 100%. The inset contains phase-contrast microscopy images showing examples of colonies formed by a Mucdhl M clone. (C) Same as in panel B with clones seeded in soft agar. (D) Mucdhl M inducible cells were grown for 7 days without doxycycline and subsequently treated or not by doxycycline. Phase-contrast microscopy images show examples of a colony formed after 11 and 21 days of culture. Bright-field microscopy images show all the colonies (paraffin embedded/H&E stained) after 21 days of culture. Original magnification, ×200, except for paraffin-embedded colonies at ×50 (bottom panels). * P < .01 and ** P < .001. Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Mucdhl can reduce tumor formation of colon cancer cells. (A) Volumes (±standard error mean) of subcutaneous tumors formed upon injection of control (no doxycycline, squares) or expressing Mucdhl M (with doxycycline, circles) HCT116 stable cells in nude mice. (B) As in panel A with a Mucdhl L clone. Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Mucdhl M can interact with β-catenin and antagonize its transcriptional activity. (A) HEK293 cells were transfected with Flag-β-catenin and HA-Mucdhl plasmids before immunoprecipitation (IP) with an anti-HA antibody. Proteins in bulk lysates (inputs, 1%) and immunoprecipitates were separated by SDS-PAGE and detected by Western blot. (B) Extracts from Caco2/TC7 grown for 2 or 17 days were incubated with IgG or an anti-β-catenin (cat) antibody for immunoprecipitation before analysis by Western blot. (C) HEK293 cells were transfected to overexpress β-catenin alone or with Flag-tagged Mucdhl M before in situ PLA using anti-Flag and anti-β-catenin antibodies. Green fluorescence corresponds to the PLA-positive signal and indicates that the 2 molecules belong to the same protein complex; blue fluorescence corresponds to nuclei (DAPI). Original magnification, ×400. (D) Stable Mucdhl M-expressing HCT116 cells were grown in soft agar for 21 days with treatment (±doxycycline) starting at day 7. Colonies were paraffin-embedded, sectioned, and immunostained. Original magnification, ×200. (E) SW480 cells were transfected with increasing doses of Mucdhl-expressing plasmid or empty plasmid (−), the Topflash reporter plasmid, and a promoter-less renilla reporter plasmid before luminescence measurement. Relative promoter activity (±standard deviation, normalized with renilla values) of the Topflash reporter in the absence of Mucdhl was set at 1. (F) As in panel E with HEK293 cells being transfected with β-catenin S33 and TCF4-expressing plasmids or empty plasmids. * P < .01 and ** P < .001. Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 6 Gastroenterology 2012 142, 875-885.e3DOI: (10.1053/j.gastro.2011.12.037) Copyright © 2012 AGA Institute Terms and Conditions