DNA demethylation caused By 5-Aza-2’-Deoxycytidine induces mitotic alterations and aneuploidy

P14^ARF is a tumor suppressor encoded by the CDKN2a locus that is frequently inactivated in human tumors. P14^ARF protein quenches oncogene stimuli by inhibiting cell cycle progression and inducing apoptosis. P14^ARF functions can be played through interactions with several proteins. However, the majority of its activities are notoriously mediated by the p53 protein. Interestingly, recent studies suggest a new role of p14^ARF in the maintenance of chromosome stability. Here, we deepened this new facet of p14^ARF which we believe is relevant to its tumor suppressive role in the cell. To this aim, we generated a monoclonal HCT116 cell line expressing the p14^ARF cDNA cloned in the piggyback vector and then induced aneuploidy by treating HCT116 cells with the CENP-E inhibitor GSK923295. P14^ARF ectopic re-expression restored the near-diploid phenotype of HCT116 cells, confirming that p14^ARF counteracts aneuploid cell generation/proliferation.

The centromere is a fundamental chromosome structure in which the macro-molecular kinetochore assembles and is bound by spindle microtubules, allowing the segregation of sister chromatids during mitosis. Any alterations in kinetochore assembly or functioning or kinetochore–microtubule attachments jeopardize chromosome stability, leading to aneuploidy, a common feature of cancer cells. The spindle assembly checkpoint (SAC) supervises this process, ensuring a faithful segregation of chromosomes. CENP-E is both a protein of the kinetochore and a crucial component of the SAC required for kinetochore–microtubule capture and stable attachment, as well as congression of chromosomes to the metaphase plate. As the function of CENP-E is restricted to mitosis, its haploinsufficiency has been used to study the induced cell aneuploidy; however, the gene expression profile triggered by CENP-E reduction in normal cells has never been explored. To fill this gap, here we investigated whether a gene network exists that is associated with an siRNA-induced 50% reduction in CENP-E and consequent aneuploidy. Gene expression microarray analyses were performed at early and late timepoints after transfection. Initially, cell cycle regulation and stress response pathways were downregulated, while afterwards pathways involved in epithelial–mesenchymal transition, hypoxia and xenobiotic metabolism were altered. Collectively, our results suggest that CENP-E reduction triggers a gene expression program that recapitulates some features of tumor cells.