Abstract 2194: p21Waf1/Cip1 dependent chromatin modifications in colorectal tumor cells

Abstract Objectives: The cell cycle regulator p21Waf1/Cip1 belongs to the family of cyclin-dependent kinase inhibitors (CDKIs). Depending on the cellular condition, p21Waf1/Cip1 can either, inhibit the cell cycle or promote proliferation, prohibit or induce apoptosis and increase or decrease cell differentiation. The contradictory nature of p21Waf1/Cip1 encouraged us to investigate the effects of this CDKI on the expression of chromatin modifying enzymes in colorectal tumor cells. Methods: Three different HCT116 cell lines were used for our studies. HCT116 WT, HCT116 p21-/- and HCT116 p53-/-. The isolated RNA was analyzed via Epigenetic Chromatin Modification Enzymes RT2 Profiler PCR Arrays from Qiagen. Western Blot analysis was performed to verify the results of the PCR arrays. In addition, immunohistochemical staining of different chromatin modifiers was performed on tissue microarrays of colon cancer patients. The protein structure of p21Waf1/Cip1 was generated by computational modelling techniques to investigate possible interactions between p21Waf1/Cip1 and chromatin modifying enzymes. Ex vivo crypt organoids from WT and p21 -/- mice were studied for chromatin signature. Results: The PCR arrays provided us with astonishing results. Compared to the wild type HCT116 cells, we could see a clear deregulation of a high number of different chromatin modifying enzymes in the two knock out cell lines. SETD1B was up-regulated 1,8 times in p21-/- and HDAC1, which is a known interaction partner of p21Waf1/Cip1, was down regulated about 2 times compared to the WT cells. This could also be proven by Western Blot and immunohistochemical analysis. The data was further processed and analyzed using an R package termed as HTqPCR and it was observed that ATF2 was up-regulated, while AURKB, different HDACs, and DNMT3B genes were down-regulated in p21-/- cells compared to the WT cells. The interaction between p21Waf1/Cip1 and HDAC1 could be analyzed on an atomic scale by molecular dynamics simulations. Conclusion: Our data strongly suggests that p21Waf1/Cip1 is very important for chromatin regulation in colon cancer cells and that its loss induces an active histone code. Interestingly most p21-dependent effects were independent of p53. These exciting and new findings have to be further investigated. Citation Format: Pablo Lennert, Katharina Erlenbach-Wuensch, Carsten Kroeger, Sara Steinmann, Kerstin Huebner, Sushmita Paul, Felix Rausch, Arndt Hartmann, Regine Schneider-Stock. p21Waf1/Cip1 dependent chromatin modifications in colorectal tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2194. doi:10.1158/1538-7445.AM2015-2194