p16INK4A is a β-catenin target gene and its expression correlates with low survival in human colorectal cacner

1259 The cell division cycle (cdc) inhibitor p16 INK4A (inhibitor of kinase 4) is over-expressed at the invasion front of many colorectal carcinomas (CRC). Here , p16 INK4A expression correlates with high statistical correlation with the nuclear expression of β-catenin. Thus, we hypothesized that p16 INK4A might be a target gene of β-catenin. The promoter/enhancer of the p16 INK4A gene harbours two consensus TCF-binding elements (TBE, WWCAAAG). The proximal TBE binds specifically recombinant glutathione-S-transferase-T-cell factor-4 (GST-TCF4) fusion protein as shown by employing electro-mobility shift assays (EMSA). Using chromatin immune-precipitations (ChIP) it was shown that TCF/β-catenin binds to the proximal TBE of the p16 INK4A gene in the cultured colorectal tumor cell line HCT116. β-catenin transactivated p16 INK4A luciferase-reporter-constructs in a dose dependent manner like ets-2 did, another p16 INK4A transactivator. The β-catenin transferred transactivation was lost, when the proximal TBE was mutated. But ets-2 still was able to transactivate. A dominant negative (dn) form of TCF was able to suppress transactivation. Together with the histone acetyl transferases (HAT) p 300 or CBP (CREB binding protein) β-catenin super-stimulated activation of p16 INK4A luciferase-reporter constructs. Using transient adenoviral transduction employing dnTCF4 encoding adenoviruses or RNA interference (RNAi) using small interfering (si) RNA specific for β-catenin it was shown that p16 INK4A was down-regulated specifically compared to the known target genes c-myc and cyclin D1 as well as several house keeping genes. Finally, in a collection of human CRC displaying an infiltrative invasion front associated with a nuclear expression of β-catenin and p16 INK4A in tumor cells at the invasion front we showed that p16 INK4A expression correlates highly significant with lower survival enrolling Kaplan-Meier statistics and the log-rank test. Taken together the expression of p16 INK4A a potent inhibitor of the cdc is regulated specifically via β-catenin in cultured colorectal tumor cell lines and correlates with lower survival in a large subset of CRCs. Thus, the importance of proliferation, which is found associated with tumor cells outside the invasion front, might have been over-estimated for the progression of CRC with an infiltrative invasion fronts. If one counts low proliferation as an indicator of adult stem cells the expression of p16 INK4A together with the expression of the two other β-catenin target genes hTERT (human telomerase RT-component) and survinin is another support of the proposed opinion that the tumor cells at the invasion front of CRC might resemble tumor stem cells. This would be functionally supported by the low survival of patients with CRCs characterized by strong p16 INK4A expression.