P3-01-01: Geminin Overexpression Prevents the Completion of Topoisomerase IIa Chromosome Decatenation Leading to Aneuploidy in Human Mammary Epithelial Cells.

Topoisomerase IIα (TopoIIα) cleaves 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 DNA end it cleaves and prevents DNA re-ligation and triggers cell death in cancer cells. However, development of resistance to these agents limits their therapeutic use. In this study, we examined therapeutic targeting of geminin for improving the therapeutic potential of TopoIIa agents. Human mammary epithelial (HME) and breast cancer cell lines were used. Geminin, TopoIIα, Cdc7 silencing was done using specific siRNAs. Transit or stable inducible overexpression of these proteins and CKI∈ were also used, as well as several pharmacological inhibitors that target TopoIIα, Cdc7, or CKI∈. We manipulated HME cells expressing H2B-GFP, in order to detect chromosome bridges. Immunoprecipitation and direct western blot were used to detect interactions between these proteins and their total expression, respectively, whereas interactions on chromosomal arms were detected using the TARDIS assay. TopoIIα phosphorylation by Cdc7 or CKI∈ was done using in vitro kinase assay. The TopoGen decatenation kit was used to measure TopoIIα decatenation activity. Finally, comet assay and metaphase chromosome spread were used to detect chromosome breakages and changes in chromosome condensation or numbers, respectively. We found that geminin and TopoIIα interact in G2/M/early G1 cells on chromosomes, that geminin recruits TopoIIα to chromosomal decatenation sites or vice versa, and that geminin silencing in HME cells triggers the formation of chromosome bridges through suppressing TopoIIα access to chromosomal arms. CKIe kinase phosphorylates and positively regulates TopoIIα chromosome localization and function. CKIe kinase overexpression or Cdc7 kinase silencing (also phosphorylates TopoIIα 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 enzymes SENP1 and SENP2 to desumoylate chromosomal bound TopoIIα and promote its release from chromosomes following completion of DNA decatenation. In cells overexpressing geminin, premature departure of TopoIIα from chromosomes is thought to be due to the fact that geminin recruits more of these desumoylating enzymes, or recruits them earlier, to chromosomal bound TopoIIα. This triggers premature release of TopoIIα from chromosomes, which we propose induces aneuploidy in HME cells, since chromosome breakages generated through were not sensed and/or repaired and the cell cycle was not arrested. TopoIIα recruitment and its chromosome decatenation function require normal level of geminin. Geminin silencing induces a cytokinetic checkpoint in which Cdc7 phosphorylates TopoIIα and inhibits its chromosomal recruitment and decatenation function. Geminin overexpression prematurely desumoylates TopoIIα, triggering its premature departure from chromosomes and leading to chromosomal abnormalities and the formation of aneuploid, drug resistant cancer cells. We propose that therapeutic targeting of geminin is essential for improving the therapeutic potential of TopoIIα agents. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-01.