Abstract 1064: Numb, a progenitor cell fate determinant and intrinsic inhibitor of Notch signaling, is cell cycle regulated and required for proper mitotic entry and progression in melanoma cells

Abstract

Abstract Proper regulation of the cell cycle, particularly DNA replication and cell division, are crucial for correct cell ploidy and genomic integrity. Loss of various regulators, often tumor suppressors, during these processes leads to either cell cycle arrest or unregulated progression through mitosis. This may result in aneuploidy and neoplastic transformation. Therefore, defining the mechanisms by which various tumor suppressors exert their regulatory functions is essential for understanding cancer development as well as developing novel target-based therapies to combat cancer progression. We found that Numb expression is cell cycle regulated and that its expression is required for proper mitotic entry and exit in melanoma cells. Using a double thymidine arrest and release protocol, we evaluated the expression of Numb during the cell cycle. Numb mRNA and protein expression was found to be maintained at a low steady state level in asynchronous A375 and HS294T melanoma cells. However, as cells entered S phase Numb levels were found to increase, peak during mitosis and then rapidly decline at mitotic exit when the cells reentered G1 Phase. This cycling of Numb mirrored the mitotic specific expression of both Plk1 and CyclinB1, however Numb showed an expression pattern with an increase beginning slightly earlier (as early as S phase). Plk1 inhibition (via shRNA as well as a small molecule inhibitor) and nocodazole-mediated mitotic arrest was also found to be accompanied with an increase in Numb expression. Additionally, we found that Plk1 and Numb co-immunoprecipitated and Plk1 was capable of phosphorylating Numb indicating a possible regulation of Numb by Plk1. Further, surprisingly, we found that lentiviral shRNA mediated knockdown of Numb resulted in a G2/M cell cycle arrest (using FACS analysis) and a significant reduction in cell growth (using Trypan blue exclusion) in both melanoma cell lines. Overall, our observations suggests that Numb is a regulator of proper cell cycle progression and may possess tumor suppressor functions. 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 1064.