Introduction

Approximately 15% of muscle invasive bladder cancers harbor mutations in the CDKN1A gene, which encodes the p53 target gene p21, with the majority of alterations truncating the peptide. We previously found that proximal truncation of p21 sensitized cells to cisplatin, while distal truncations made them more resistant. We hypothesized that truncations preserving the CDKi domain but ablating the PCNA binding domain would abrogate p21's ability to sequester PCNA but maintain cell cycle arrest capacities, enhancing the interaction between mono-ubiquitiniated-PCNA (ub-PCNA) and DNA polymerase eta (Pol η), resulting in increased TLS and DNA damage tolerance.

Methods

We used SW780 cell line for this study based on its resistance to cisplatin and p53 and p21 WT status. CRISPR sgRNAs designed to target amino acids 12 and 109 (SW780-sg12 and SW780-sg109) were used to introduce frameshifts and disrupt the majority of the peptide or only the PCNA the binding domain, respectively. sgRNA designed to target GFP was used as a control (sgGFP). We treated the cells with GI50 dose of cisplatin and analyzed post-cisplatin cell-cycle profile over 96 hours using PI-FACS and immunoblot. We treated all three cell types with increasing doses (0-40 μM) of cisplatin for 24 hours and measured the levels of ub-PCNA (K164) and Pol η using immunoblot.

Results

SW780-sgGFP arrested in S-phase at 48 and 72 hours but overcame this arrest at 96 hours and began to cycle again. SW780-sg12 were unable to arrest in S-phase, whereas SW780-sg109 were able to arrest in S-phase at 48 to 96 hours. This pattern of S-phase arrest was further confirmed by prolonged upregulation of cyclin D1 in SW780-sg109 and brief upregulation in SW780-sgGFP. ub-PCNA and Pol η accumulated in a dose responsive manner in all three cell types at 24 hours after cisplatin treatment. ub-PCNA increased abruptly at higher cisplatin doses in SW780-sgGFP, whereas the increase was gradual in CDKN1A-edited cells. Interestingly, in SW780-sg109, Pol η induction was strongest at all doses.

Conclusion

Cisplatin causes S-phase arrest in SW780-sgGFP cells, possibly by inducing replicative stress. However, this arrest is eventually bypassed and the cells progress through the cell cycle. S-phase arrest is not possible in the absence of a functioning p21, indicating that p21 plays a vital role in bringing about post-cisplatin cell cycle arrest. Interestingly, truncated p21 with intact CDKi domain is capable of causing stronger and more prolonged S-phase arrest compared to the full-length peptide. TLS may play a role in DNA damage tolerance in SW780 cells as indicated by elevated ub-PCNA and pol η. Stronger accumulation of Pol η in SW780-sg109, possibly enhances TLS and thus cisplatin resistance. Moving forward, we would like to confirm the anticipated competition between p21 and Pol η for PCNA and further verify this phenomenon in other bladder cancer cell lines.

Citation Format: Rahmat K. Sikder, Wafik S. El-Deiry, Philip H. Abbosh. Differential effects of N-terminal vs C-terminal truncating CDKN1A mutations on cisplatin resistance in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2307.