Novel Roles for P53 in the Genesis and Targeting of Tetraploid Cancer Cells

Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the “tetraploidy checkpoint”, p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).     

Two 4N Cell-Cycle Arrests Contribute to Cisplatin-Resistance

Cisplatin is a platinum-based drug that is used for the treatment of a wide-variety of primary human cancers. However, the therapeutic efficacy of cisplatin is often limited by intrinsic or acquired drug resistance. An important goal, therefore, is to identify mechanisms that lead to cisplatin resistance in cancer, and then use this information to more effectively target resistant cells. Cisplatin-resistant clones of the HCT116 cell line underwent a prolonged G2 arrest after cisplatin treatment while sensitive clones did not. The staurosporine analog UCN-01 abrogated this G2 arrest and sensitized the resistant clones to cisplatin. At later time points, 4N arrested cells assumed a tetraploid G1 state that was characterized by depletion of Cyclin A, Cyclin B, and CDC2, and increased expression of p53 and p21, in 4N cells. siRNA-mediated knockdown of p21 abrogated the tetraploid G1 arrest and induced killing that was dependent on p53. The results identify two targetable 4N arrests that can contribute to cisplatin resistance: First, a prolonged G2 arrest that can be targeted by UCN-01, and second, a tetraploid G1 arrest that can be targeted by siRNA against p21.