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

Date Published: November 7, 2014

Publisher: Public Library of Science

Author(s): Batzaya Davaadelger, Hong Shen, Carl G. Maki, Andrei L. Gartel.

http://doi.org/10.1371/journal.pone.0110844

Abstract

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).

Partial Text

Tetraploid cells contain twice the normal amount of DNA and are rare in most normal tissues. However, tetraploid cells are relatively common in cancer and are thought to contribute to tumor development, aneuploidy, and therapy resistance [1]. Direct evidence for the tumorigenic potential of tetraploid cells was provided by Fujiwara et al. [2] who isolated binucleated, tetraploid mammary epithelial cells from p53-null mice. Remarkably, these cells were more susceptible to carcinogen-induced transformation (soft-agar growth) than diploid counterparts, and the tetraploid cells formed tumors in nude mice while diploid cells did not. Other studies have linked tetraploidy to radiation and chemotherapy resistance. For example, Castedo et al. [3], [4] isolated tetraploid and diploid clones from two human cancer cell lines with wild-type p53. Importantly, tetraploid clones were resistant to radiation and multiple chemotherapy agents compared to diploid counterparts. Finally, there is mounting evidence that aneuploid cancer cells are generated from either asymmetric division or progressive chromosomal loss from tetraploid precursors. Early evidence for this came from studies in premalignant Barrett’s esophagus. In these studies, the appearance of tetraploid cells correlated with p53 loss and preceded gross aneuploidy and carcinogenesis [5], [6]. In sum, tetraploid cells can have higher tumorigenic potential, be therapy and radiation-resistant, and be precursors to cancer aneuploidy. It is therefore important to identify how tetraploid cells arise and how they can be targeted for cancer treatment.

Tetraploid (4N) cells contain twice the DNA content of diploid (2N) cells. Tetraploid 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 [1]–[6]. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. Nutlin-3a (Nutlin) is a small molecule MDM2 antagonist and activator of p53. In our previous studies, transient (24 hr) Nutlin treatment promoted a tetraploid G1 arrest in multiple cancer cell lines that express wild-type p53 [18], [19]. This tetraploid G1 arrest was characterized by depletion of G2/M proteins (e.g. Cyclins A/B, CDC2) and increased expression of G1-arrest proteins (e.g. p53, p21) in 4N cells. Upon Nutlin removal, 4N cells reinitiated DNA synthesis and replicated their DNA without an intervening mitosis, a process known as endoreduplication [18], [19]. Both the tetraploid G1 arrest and endoreduplication after Nutlin removal were dependent on p53 and p21. Finally, stable tetraploid clones could be isolated from Nutlin treated cells, and these cells were resistant to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis [19]. These findings suggested transient p53 activation by Nutlin can promote endoreduplication and the generation of therapy resistant tetraploid cells. However, a caveat is that these previous studies were done in cancer cell lines, which could include a mixture of diploid and tetraploid cells, some of which may be inherently IR/CP-resistant. It is possible that we had isolated IR/CP-resistant tetraploid clones that were already present in the population, or that the tetraploid clones we isolated were derived from individual diploid cells that were already IR/CP-resistant. Therefore, in the current report we asked if Nutlin could promote tetraploid cell formation from diploid precursors and, if yes, whether the resulting tetraploid cells would show increased resistance to IR and/or CP. Individual diploid clones (D3, D8) isolated from the HCT116 colon cancer cell line by limiting dilution were treated with Nutlin for 24 hrs, followed by Nutlin removal. Nutlin promoted a tetraploid G1 arrest in the diploid clones that was p53-dependent. The clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to IR/CP-induced apoptosis compared their diploid counterparts. 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, tetraploid clones selected after Nutlin treatment expressed twice as much P53 and MDM2 mRNA as diploid cells, expressed 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 a role 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).

Transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells. 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).

 

Source:

http://doi.org/10.1371/journal.pone.0110844