4.5 Article

Geminin overexpression prevents the completion of topoisomerase II alpha chromosome decatenation, leading to aneuploidy in human mammary epithelial cells

Journal

BREAST CANCER RESEARCH
Volume 13, Issue 3, Pages -

Publisher

BIOMED CENTRAL LTD
DOI: 10.1186/bcr2884

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Funding

  1. American Cancer Society [118046-RSG-09-275-01-CSM]

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Introduction: The nuclear enzyme topoisomerase II alpha (TopoII alpha) is able to cleave DNA in a reversible manner, making it a valuable target for agents such as etoposide that trap the enzyme in a covalent bond with the 5' DNA end to which it cleaves. This prevents DNA religation and triggers cell death in cancer cells. However, development of resistance to these agents limits their therapeutic use. In this study, we examined the therapeutic targeting of geminin for improving the therapeutic potential of TopoII alpha agents. Methods: Human mammary epithelial (HME) cells and several breast cancer cell lines were used in this study. Geminin, TopoII alpha and cell division cycle 7 (Cdc7) silencing were done using specific small interfering RNA. Transit or stable inducible overexpression of these proteins and casein kinase I epsilon (CKI epsilon) were also used, as well as several pharmacological inhibitors that target TopoII alpha, Cdc7 or CKI epsilon. We manipulated HME cells that expressed H2B-GFP, or did not, to detect chromosome bridges. Immunoprecipitation and direct Western blot analysis were used to detect interactions between these proteins and their total expression, respectively, whereas interactions on chromosomal arms were detected using a trapped in agarose DNA immunostaining assay. TopoII alpha phosphorylation by Cdc7 or CKI epsilon was done using an in vitro kinase assay. The TopoGen decatenation kit was used to measure TopoII alpha decatenation activity. Finally, a comet assay and metaphase chromosome spread were used to detect chromosome breakage and changes in chromosome condensation or numbers, respectively. Results: We found that geminin and TopoII alpha interact primarily in G(2)/M/early G(1) cells on chromosomes, that geminin recruits TopoII alpha to chromosomal decatenation sites or vice versa and that geminin silencing in HME cells triggers the formation of chromosome bridges by suppressing TopoII alpha access to chromosomal arms. CKI epsilon kinase phosphorylates and positively regulates TopoII alpha chromosome localization and function. CKI epsilon kinase overexpression or Cdc7 kinase silencing, which we show phosphorylates TopoII alpha in vitro, restored DNA decatenation and chromosome segregation in geminin-silenced cells before triggering cell death. In vivo, at normal concentration, geminin recruits the deSUMOylating sentrin-specific proteases SENP1 and SENP2 enzymes to deSUMOylate chromosome-bound TopoII alpha and promote its release from chromosomes following completion of DNA decatenation. In cells overexpressing geminin, premature departure of TopoII alpha from chromosomes is thought to be due to the fact that geminin recruits more of these deSUMOylating enzymes, or recruits them earlier, to bound TopoII alpha. This triggers premature release of TopoII alpha from chromosomes, which we propose induces aneuploidy in HME cells, since chromosome breakage generated through this mechanism were not sensed and/or repaired and the cell cycle was not arrested. Expression of mitosis-inducing proteins such as cyclin A and cell division kinase 1 was also increased in these cells because of the overexpression of geminin. Conclusions: TopoII alpha recruitment and its chromosome decatenation function require a normal level of geminin. Geminin silencing induces a cytokinetic checkpoint in which Cdc7 phosphorylates TopoII alpha and inhibits its chromosomal recruitment and decatenation and/or segregation function. Geminin overexpression prematurely deSUMOylates TopoII alpha, triggering its premature departure from chromosomes and leading to chromosomal abnormalities and the formation of aneuploid, drug-resistant cancer cells. On the basis of our findings, we propose that therapeutic targeting of geminin is essential for improving the therapeutic potential of TopoII alpha agents.

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