Muscle-invasive bladder cancer (MIBC) frequently harbors mutations in the CDKN1A gene, which encodes the tumor suppressor protein p21, with the majority of alterations truncating the peptide. The effect of these mutations is poorly understood. We hypothesized that after DNA-damaging events, cells deficient in p21 would be unable to halt the cell-cycle and efficiently repair DNA damage, thus proceeding down the apoptotic pathway. We used synthetic CRISPR guide RNAs to ablate the whole peptide (sg12, targeting the 12th amino acid) or the C-terminal Proliferating Cell Nuclear Antigen (PCNA)-binding domain (sg109) to mimic different p21-truncating mutations compared to a negative control (sgGFP) in bladder cancer cell lines. Loss of detectable p21 and a stable truncated p21 peptide were identified in sg12 and sg109 single cell clones, respectively. We found that p21-deficient cells (sg12) were sensitized to cisplatin, while cells harboring distally truncated p21 (sg109 clones) demonstrated enhanced cisplatin resistance. p21-deficient sg12 clones demonstrated less repair of DNA-platinum adducts and increased -H2AX foci after cisplatin exposure, suggesting there was persistent DNA damage after p21 loss. p21-deficient sg12 clones were also unable to prevent the activation of CDK1 after DNA damage, and therefore, continued through the cell cycle, resulting in replication fork collapse, potentially explaining the observed cisplatin sensitization. sg109 clones were neither unable to sequester PCNA nor localize to the nucleus after DNA damage, potentially explaining the chemoresistant phenotype. Our findings suggest that different CDKN1A truncations have different and perhaps disparate biology, and that there may be a duality of effect on cisplatin sensitivity depending on mutation context. Implications: Truncating CDKN1A mutations generate a retained peptide that may have neomorphic functions and impact cisplatin sensitivity in patients with bladder cancer.

中文翻译：

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临床相关 N- 与 C- 末端截断 CDKN1A 突变对膀胱癌顺铂敏感性的不同影响

肌肉浸润性膀胱癌 (MIBC) 经常在 CDKN1A 基因中发生突变，该基因编码肿瘤抑制蛋白 p21，大部分改变会截断该肽。这些突变的影响知之甚少。我们假设在 DNA 损伤事件后，缺乏 p21 的细胞将无法停止细胞周期并有效修复 DNA 损伤，从而沿着凋亡途径进行。我们使用合成的 CRISPR 指导 RNA 来消融整个肽（sg12，靶向第 12 个氨基酸）或 C 末端增殖细胞核抗原 (PCNA) 结合域 (sg109)，以模拟与阴性对照相比不同的 p21 截断突变(sgGFP) 在膀胱癌细胞系中。分别在 sg12 和 sg109 单细胞克隆中鉴定出可检测的 p21 缺失和稳定的截短 p21 肽。我们发现 p21 缺陷细胞 (sg12) 对顺铂敏感，而含有远端截短 p21 的细胞 (sg109 克隆) 表现出增强的顺铂抗性。p21 缺陷 sg12 克隆在顺铂暴露后表现出较少的 DNA-铂加合物修复和增加的 -H2AX 病灶，这表明在 p21 丢失后存在持续的 DNA 损伤。p21 缺陷型 sg12 克隆也无法在 DNA 损伤后阻止 CDK1 的激活，因此，在整个细胞周期中持续存在，导致复制叉塌陷，这可能解释了观察到的顺铂致敏。sg109 克隆既不能隔离 PCNA，也不能在 DNA 损伤后定位到细胞核，这可能解释了化学抗性表型。我们的研究结果表明，不同的 CDKN1A 截断具有不同且可能不同的生物学，并且取决于突变背景，对顺铂敏感性可能存在双重影响。启示：截断 CDKN1A 突变会产生一种保留肽，该肽可能具有新形态功能并影响膀胱癌患者对顺铂的敏感性。