Abstract 42: The NF-κB/MDM2 pathway is engaged in activin/ PI3 kinase induced degradation of p21 in colon cancer cells

Introduction: The signaling pathways for activin and TGFβ are frequently disrupted in colorectal cancers (CRC) and these mutations are associated with metastatic disease. Understanding the cellular consequences wrought by mutations to these signaling pathways may provide opportunities for the development of new therapeutic approaches. Our lab has previously shown opposing effects on a downstream target of TGFβ and activin, namely the cyclin-dependent kinase (CDK) inhibitor p21. TGFβ induces SMAD4-dependent upregulation of p21, while activin downregulates p21 in a SMAD4-independent mechanism via ubiquitination and degradation allowing for enhanced cell migration. However, the mode of regulation leading to p21 degradation is not completely known. We hypothesize that regulation of the E3 ubiquitin ligase MDM2 by NF-κB is involved in p21 degradation. Here we explore the role of MDM2 in the regulation of p21 in response to activin and TGFβ treatment in FET CRC cells. Methods: Colon cancer cell lines were assessed for ligand-induced PI3K/NF-κB/MDM2/p21 pathway engagement. FET cells were transfected with increasing concentrations of either wild type p65 or a dominant-negative mutant of p65 and treated with activin or TGFβ prior to immunoblot analysis for IκB-alpha, MDM2 and p21. DNA-binding activity of NF-κB was assessed by electrophoretic mobility shift assay (EMSA). The requirement for NF-κB activation was determined through use of the selective inhibitor peptide NBD (NEMO-binding domain), and PI3 kinase activity was confirmed by inhibition with LY294002. Results: Because activin induces ubiquitination of p21, we investigated the potential involvement of MDM2 and further the regulation of MDM2 by NF-κB. NF-κB has been reported to regulate MDM2 through direct binding to an NF-κB binding site in the promoter of MDM2. Activin induced activation of NF-κB as measured by increased DNA binding activity to an NF-κB consensus-binding site as well as by a reduction in IκB-alpha protein. Furthermore, we found this regulation to be dependent on activation of PI3 kinase. Overexpression of wild type p65 plasmid but not dominant negative p65 was found to increase MDM2 expression while the NF-κB specific inhibitor NBD blocked the activin-induced increase in MDM2. By contrast, TGFβ treatment did not contribute to an increase in MDM2 levels via NF-κB pathway. Conclusions: Here, we provide evidence to indicate that in colon cancer cells, activin may be working through NF-κB to promote MDM2 activity leading to the degradation of p21 in a PI3 kinase dependent mechanism. Future experiments will explore if pharmaceutical intervention in this pathway can inhibit the metastatic activity of activin as a therapeutic approach for advanced colon cancer. Citation Format: Arundhati Jana, Seung Hyua Baik, Timothy Carroll, Ozkan Ozden, Nancy L. Krett, Barbara Jung. The NF-κB/MDM2 pathway is engaged in activin/ PI3 kinase induced degradation of p21 in colon cancer 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 42. doi:10.1158/1538-7445.AM2015-42