Abstract 1062: Numb, an intrinsic Notch inhibitor, possesses a tumor suppressor role through regulation of Plk1 localization during mitosis

Abstract

Numb, an intrinsic Notch inhibitor and cell fate determinant, has been shown to possess many functions in proper cell division. These include its role in endocytosis, a tumor suppressor role via regulation of p53 stability, and asymmetric self renewal during progenitor cell division. Studies have shown that a proper localization of fully functional Plk1 is required for mitotic entry, progression and exit. Plk1 has been shown to play a critical role in bi-polar spindle formation and maturation during mitosis. It has been shown that chemical inhibition as well as genetic knockdown of Plk1 results in cells forming a mono-polar spindle phenotype surrounded by DNA rather than a bi-polar arrangement on either side of the metaphase plate. This severe mitotic phenotype results in a G₂/M cell cycle arrest and mitotic catastrophe. Also, milder inhibition of Plk1 has been shown to result in errors in bi-polar spindle formation including multi-polar phenotypes or incomplete microtubule attachment and division. Some of these less pronounced phenotypes may not prevent cell division and may contribute to cellular aneuploidy and cancer. In this study, employing melanoma cell lines A375 and HS294T, we demonstrated that Numb and Plk1 interact as shown by co-immunoprecipitation and immunofluorescent assays. In addition, we found that both Plk1 and Numb co-localize to the spindle poles in metaphase. Further, using lentiviral shRNA knockdown of Plk1 or Numb, we also found that loss of Plk1 or Numb dysregulates the localization of the other. Most striking was the pronounced mislocalization of Plk1 from the spindle poles. Although, the majority of metaphase cells were still bi-polar, there was a marked increase in mislocalized γ-tubulin in some instances. Additionally, in the cells with two defined spindle poles, γ-tubulin still appears to be less pronounced at strictly the spindle pole with an apparent indistinct staining surrounding the poles indicating that Numb may not be directly required for γ-tubulin localization to the spindles but the misregulated Plk1 localization slows or prevents spindle pole maturation and γ-tubulin recruitment. Conversely, the differing phenotypes of γ-tubulin localization may also be explained by a redundant role of Numb-like, though this is yet to be explored. Overall, these data suggested that Numb may possess tumor suppressor functions by regulating proper Plk1 localization and proper spindle pole maturation.

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 1062.