Abstract LB-124: RHAMM acts near chromosomes to promote mitotic spindle assembly by regulating the location and abundance of TPX2 and the downstream activation of Aurora A.

Enhanced cell proliferation is a hallmark of cancer. Thus, molecular pathways underlying mitosis are important targets for therapy. To form a bipolar mitotic spindle, the dividing cell must assemble microtubules at centrosomes [spindle poles] as well as non-centrosome sites [kinetochores]. The spindle then captures and aligns the duplicated chromosomes. Correct attachment satisfies the spindle assembly checkpoint (SAC) and allows for mitotic progression. Aurora kinase A, when bound by its activator, TPX2, is a key regulator of spindle assembly. Upon activation, the kinase phosphorylates substrates to target them to the centrosomes, and protects them from proteolytic degradation. Since it is necessary for optimal Aurora A function, TPX2 availability is tightly controlled. The receptor for hyaluronan-mediated motility (RHAMM) is a microtubule associated protein that interacts with TPX2 and participates in spindle assembly. However, it is unclear whether or how the RHAMM-TPX2 complex modulates Aurora A activity. We hypothesize that RHAMM interacts with TPX2 to determine TPX2 location and abundance, which facilitates Aurora A activity and spindle assembly. We have followed the location and interactions of RHAMM through mitosis, as well as the consequences for its loss of function. We tracked the location of RHAMM and GFP-RHAMM and found it at the kinetochores. Then, we colocalized RHAMM with key kinetochore proteins, BUBR1 and NDC80, during microtubule nucleation and confirmed physical associations by IP. For insights into the function of RHAMM, we followed mitotic cells in real-time. In HeLa cells treated with siRNA targeting RHAMM, we found a significant increase in aberrant spindle morphology and mitotic failure, and we were able to rescue these phenotypes by reintroducing GFP-RHAMM. We also measured the duration of spindle assembly, chromosome congression and cytokinesis. RHAMM depleted mitotic cells present two distinct phenotypes, and these cells either fail spindle assembly or construct phenotypically “normal” mitotic spindles that do not satisfy the SAC. Thus, the protein9s location, interactions, and the consequences of its depletion all point to a critical role at the kinetochore. Previous reports suggest RHAMM participates in spindle assembly by interacting with TPX2, however, the exact mechanism remains unknown. We find that RHAMM depletion decreases TPX2 protein abundance during early mitosis, and impairs TPX2 transit to the spindle poles, which results in reduced Aurora A protein abundance and activity. Together, these results suggest that RHAMM influences Aurora A actions by regulating the stability and location of TPX2. Further insights into RHAMM associated pathways may lead to the discovery of new therapeutic targets and optimize the usage of promising drugs such as Aurora A inhibitors. Citation Format: Helen Chen, Chinten Lim, Chris Maxwell. RHAMM acts near chromosomes to promote mitotic spindle assembly by regulating the location and abundance of TPX2 and the downstream activation of Aurora A. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-124. doi:10.1158/1538-7445.AM2013-LB-124