Abstract PR13: Germ-line mutations in CDC20 result in familial cancers via deregulation of the cell cycle
Authors: Castellsague Ester , Carrot-Zhang Jian , Gamache Isabelle , Rivera Barbara , Moustafa Mohamed , Barford David , Majewski Jacek , Teodoro Jose , Foulkes William David 
Published: November 2016

Introduction: Cdc20 is a co-activator recruited by the anaphase-promoting complex (APC), a large, multi-subunit complex that functions as an Ubiqutin ligase that targets key substrates for degradation to promote cell cycle progression. During mitosis the APC/Cdc20 complex is inhibited by the spindle assembly checkpoint (SAC) pathway, which ensures the correct chromosome segregation during mitosis. When associated with the mitotic checkpoint complex (MCC), formed by Mad2, BubR1 and Bub3 proteins, Cdc20 cannot activate the APC and cell cycle is arrested. Deregulation of this pathway can result in cells proceeding through mitosis before proper chromosomal attachment to the kinetochore, potentially leading to aneuploidy and cancer.

We have found 2 different missense variants in CDC20 (L151R and N331K), in the affected members of two families with malignant ovarian germ cell tumors, a rare non-epithelial neoplastic transformation of ovarian primitive oocyte. Interestingly, several members of L151R family presented with other additional tumors such as colorectal or breast cancers.

Purpose: We aim to elucidate the mechanism by which L151R and N331K are able to trigger tumorigenesis, and demonstrate that the disruption of Cdc20 may play an important role in cancer.

Methods: The effect of the mutations has been measured through three different cell types: S. cerevisiae carrying the Cdc20N407K (N331K in humans); HeLa cells transfected with Cdc20L151R and Cdc20N331K; and human primary skin fibroblasts from two siblings carrying N331K mutation. Cell cycle progression has been measured in the presence of the spindle poison nocodazole by several methods: checkpoint cell survival assay, western blot of MCC and APC proteins, flow cytometer and time-lapse. The effect of the mutations on the interaction of Cdc20 and its MCC and APC partners has been assessed by immunoprecipitation.

Results: S. cerevisiae carrying the Cdc20N407K (N331K in humans) show a clear reduction in viability of the mutant compared to wild-type (wt) after the addition of nocodazole, as a result of mutant cells keep dividing despite microtubule-kinetochore attachment being destabilized, thus causing genomic instability and catastrophe in progeny cells.

HeLa cells transfected with Cdc20L151R and Cdc20N331K show a significant increase in the non-phosphorylated forms of Cdc27 and Bubr1 compared to the wt and the empty vector, indicating the abundance of non-mitotic cells increases with the mutated forms of Cdc20. The increased number of polyploidy cells (8n) in the mutants seen by flow cytometry proves that the non-mitotic cells are a result of the mutated Cdc20 overriding mitosis even in the presence of nocodazole. Additionally, time-lapse results fit with a reduced time spent in mitosis by the HeLa cells carrying the mutated forms of Cdc20. Immunoprecipitation of the transfected Cdc20 showed a reduced binding of the mutated proteins to Bubr1, suggesting that the mutated forms of Cdc20 could be over-activating the APC thanks to a reduced affinity with its MCC partner Bubr1.

Time-lapse experiments performed in human skin fibroblasts from two patients carrying the N331K mutation in heterozygosity showed similar results in terms of the length of the mitotic arrest in the presence of nocodazole.

Last but not least, we are expecting to be able to see the translation of these in vitro functional results at the tumoral level after the WES and cytogenetic analysis of the patient's tumors is finalized.

Conclusions: Our preliminary data suggest that L151R and N331K may drive cancer through the insensitivity of Cdc20 to the SAC. To our knowledge this is the first description of CDC20 as a cancer predisposition gene. The rapid division of cancer cells demands that they maintain high APC activity and this may represent an Achilles heel that can be exploited therapeutically.

This abstract is also being presented as Poster B25.

Citation Format: Ester Castellsague, Jian Carrot-Zhang, Isabelle Gamache, Barbara Rivera, Mohamed Moustafa, David Barford, Jacek Majewski, Jose Teodoro, William David Foulkes. Germ-line mutations in CDC20 result in familial cancers via deregulation of the cell cycle. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR13.