Background. Recent studies have suggested that the ability of Ras to transform cells depends on cooperation with the Notch signaling pathway. However, the molecular and cellular mechanisms involved in this cooperation remain unknown. Aim. To evaluate the impact of the Mek/Erk pathway, downstream of Ras, on Notch signaling. Methods. HEK293T and human pancreatic cancer cells MIA PaCa-2 were used. Mek/Erk activity was down-regulated by treatment with the specific inhibitor U0126 (10 µM). Transfections or retroviral infections of a constitutive active form of KRas or Mek (MekCA) were used to increase Mek/Erk activity. NIC-dependent transcription was measured by luciferase assays using a specific (CSL) luciferase reporter gene. Results. 1- Transient transfection of an activated KRas stimulated by 1.7-fold NIC-dependent transcription. Transfection of MekCA increased by 4.3-fold NIC-dependent transcription while a wild-type form of Mek (MekWT) was without effect. In parallel, treatment with U0126 reduced by 60% the basal NIC-dependent transcription and completely blocked the KRas- and MekCA-induced NIC-dependent transcription. This negative effect of U0126 on NIC-dependent transcription correlated with decreased nuclear staining of the NIC target gene Hes1. 2- Western blot analysis revealed higher molecular weight forms of NIC when cells were transfected/infected with MekCA as compared to control MekWT-transfected/infected cells. U0126 treatment prevented the MekCA-induced decreased in mobility shift of NIC suggesting that NIC is subjected to Mek-dependent post-translational modifications in vivo. 3- Immunoprecipitation followed by in vitro phosphatase assay revealed that these MekCA-induced higher molecular weight forms of NIC were phosphorylated forms of NIC. 4- By in vitro kinase assays, we demonstrated that NIC worked as Erk substrate. 5- Finally, following in vivo phospholabeling, we observed that NIC immunoprecipitated from MekCA-transfected cells was more phosphorylated as compared to NIC immunoprecipitated from MekWT-transfected cells. Conclusion. Our results provide the novel discovery that Mek/Erk signaling promotes NIC phosphorylation which correlates with increased NIC-dependent transcription. Our discovery thus provides a possible cellular mechanism by which Ras and Notch cooperate to promote cellular transformation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1234.