Volume 136, Issue 1, Pages 196-205.e2 (January 2009) p16INK4a Is a β-Catenin Target Gene and Indicates Low Survival in Human Colorectal Tumors  Stella Wassermann, Silvio K. Scheel, Elke Hiendlmeyer, Richard Palmqvist, David Horst, Falk Hlubek, Angela Haynl, Lydia Kriegl, Simone Reu, Susanne Merkel, Thomas Brabletz, Thomas Kirchner, Andreas Jung  Gastroenterology  Volume 136, Issue 1, Pages 196-205.e2 (January 2009) DOI: 10.1053/j.gastro.2008.09.019 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 The proximal TCF binding element (TBE) of the human p16INK4A promoter/enhancer binds recombinant TCF4 protein. (A) The promoter/enhancer region of the human p16INK4A gene (accession no. AF022809). The start of translation (ATG) is 1. The human p16INK4A promoter/enhancer harbors 2 consensus TBEs (WWCAAAG)18 embedded in the context of a variety of other transcription factors known to be involved in the transcriptional regulation of the p16INK4A gene: ITSE (INK4a transcription silence element),29 SP1,30 Ets,21 RBRE (retinoblastoma-responsive element),31 and RHA (RNA helicase A).32 (B) EMSA. Radioactively labeled oligonucleotides containing the proximal TBE bind specifically to the DNA-binding domain of a recombinant GST-TCF4 (G∼T) fusion protein (lane 2) but not when the TBE is mutated (lane 7). Binding is compatible using WT p16INK4A (lane 3) or c-MYC (lane 5) but not MU p16INK4A (lane 4) TBE-containing oligonucleotides. Binding does not depend on GST (lanes 6 and 9). Interaction with the radioactively labeled TBE219 of the c-MYC gene was provided as the positive control. Gastroenterology 2009 136, 196-205.e2DOI: (10.1053/j.gastro.2008.09.019) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 β-catenin binds to the proximal TBE of the human p16INK4A gene in the context of native chromatin. ChIP using antibodies specific for β-catenin, RNA polymerase II, or immunoglobulins G (IgG) as a control. Human genomic DNA (input) was used as a positive control and water as the negative control (Ø). PCR primers covered sequences of the human p16INK4A-gene containing the proximal TBE (A), the c-MYC gene containing TBE219 (B), or the GAPDH-gene harboring the TATA box (C). Intron 25 of the CNAPI-gene (D) is not expected to bind DNA and was thus used as a control for comparing the unspecific binding of DNA to the antibodies in use. β-catenin−specific antibodies precipitated DNA sequences containing TBEs of the p16INK4A- and c-myc genes (A and B) but not GAPDH gene (C). The situation was expectedly found vice versa for an antibody specific for RNA polymerase II. The prepared chromatin did not bind unspecifically to immunoglobulins (IgG), and the antibodies in use did not differ in their unspecific binding to chromatin (D). Gastroenterology 2009 136, 196-205.e2DOI: (10.1053/j.gastro.2008.09.019) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 The proximal TBE of the human p16INK4A promoter/enhancer confers β-catenin/TCF-specific transactivation. (A) After transfection into 293 cells, luciferase reporter constructs containing the WT p16INK4A promoter/enhancer (black bars) were transactivated in a dose-dependent manner by a stabilized form of β-catenin (β-cat) as well as Ets2 (ets). Together, β-catenin and Ets2 (β-cat + ets) stimulated transactivation in an additive manner. Luciferase reporter constructs containing the MU p16INK4A promoter/enhancer (stippled bars) were expectedly transactivated only by Ets2 but no longer by β-catenin. When added together, the effect was thus comparable to the activity induced by Ets2 alone. (B) In HCT116 cells, dnTCF4 suppressed the activity of luciferase reporter constructs containing the p16INK4A promoter/enhancer containing a WT but not MU TBE sequence. (C) Moreover, dnTCF4 as well as dnEts2 suppressed the transcriptional activity of WT p16INK4A in HCT116 cells. (D) Two hundred ninety-3 cells displayed an activation of human p16INK4A promoter/enhancer luciferase reporter constructs by HATs, which was synergistically stimulated by β-catenin. Gastroenterology 2009 136, 196-205.e2DOI: (10.1053/j.gastro.2008.09.019) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 β-catenin/TCF regulates the endogenous expression of p16INK4A. (A and D) dnTCF4 (dnT) in contrast to β-galactosidase (β-G) expressing adenovirus leads to the down-regulation of p16INK4A (70%) and (B and D) c-myc mRNA levels in the human colorectal cell line HCT116. (C and D) In contrast, mRNA levels of the housekeeping gene GAPDH are unaffected by dnTCF4. (D) Densitometric analysis of the band intensities displayed in the gels. (M), 100-bp marker (Invitrogen); (Ø), negative control using water instead of template. The number of cycles of the respective PCR is given below each gel photograph. (E and F) β-catenin−specific siRNA leads to the down-regulation of β-catenin-, c-MYC-, Cyclin D1- and p16INK4A-specific mRNA levels compared with the geometric mean of the 3 housekeeping genes HPRT, β-actin, and YWMAZ (geo-HKG) in the human colorectal tumor cell lines HCT116 (E) and LS174T (F). Gastroenterology 2009 136, 196-205.e2DOI: (10.1053/j.gastro.2008.09.019) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 High expression of p16INK4A at the invasive front of CRC correlates with low survival rates. Representative (A) overview (50x) and high-power magnification (250x) of (B) central areas or (C) the invasive front of a typical CRC stained immunohistochemically using p16INK4A-specific antibodies. (D) Kaplan–Meier curve and log-rank test assign high labeling index of p16INK4A to a lower survival rate in contrast to cases with a low labeling index. Gastroenterology 2009 136, 196-205.e2DOI: (10.1053/j.gastro.2008.09.019) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure Gastroenterology 2009 136, 196-205.e2DOI: (10.1053/j.gastro.2008.09.019) Copyright © 2009 AGA Institute Terms and Conditions