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Abstract：
The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo.
To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models.
Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner.
The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.
To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models.
Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner.
The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.
摘要：
背景：细胞肿瘤蛋白p53（TP53）是一种肿瘤抑制基因，在人类癌症中经常发生突变。在各种癌症类型中，非常侵袭性的高级别浆液性卵巢癌（HGSOC）表现出TP53突变的最高患病率，>96%的病例。尽管努力重新激活p53，但尚未批准临床药物来挽救p53功能。在这项研究中,我们的主要目标是将体外转录（IVT）野生型（WT）p53-mRNA施用于HGSOC细胞系，原代细胞,和原位小鼠模型，为了探索其对抑制肿瘤生长和扩散的影响，在体外和体内。
方法:为了恢复p53的活性,使用哺乳动物载体系统在HGSOC细胞系中外源表达WTp53。此外，使用脂质体将IVTWTp53mRNA递送到不同的HGSOC模型系统（原代细胞和患者来源的类器官）中，并研究其增殖，细胞周期进程，凋亡，菌落形成,和染色体不稳定。使用OVCAR-8和原代HGSOC细胞中的RNA测序分析p53mRNA诱导的转录组改变，其次是独创性途径分析。使用原位异种移植物和转移性腹膜内小鼠模型研究了对肿瘤生长和转移的体内作用。
结果：使用新设计的基于IVTmRNA的方法，在不同的HGSOC模型系统中探索了TP53抑癌基因的再激活。WTp53mRNA的引入引发剂量依赖性细胞凋亡，细胞周期停滞,和有效的持久抑制HGSOC细胞增殖。基于mRNA的p53再激活后OVCAR-8细胞的转录组分析揭示了与p53信号相关的基因表达的显著改变。如细胞凋亡，细胞周期调节，和DNA损伤。同时恢复p53功能可减少HGSOC细胞内的染色体不稳定性，强调其在保护基因组完整性方面的关键贡献，通过缓和由复制应激引起的双链断裂的基线发生。此外，在各种老鼠模型中，p53mRNA治疗以剂量依赖性方式减少了肿瘤生长并抑制了肿瘤细胞在腹膜腔中的传播。
结论：基于IVTmRNA的p53再激活有望成为HGSOC的潜在治疗策略，为p53功能的分子机制及其在卵巢癌治疗中的相关性提供有价值的见解。
方法:为了恢复p53的活性,使用哺乳动物载体系统在HGSOC细胞系中外源表达WTp53。此外，使用脂质体将IVTWTp53mRNA递送到不同的HGSOC模型系统（原代细胞和患者来源的类器官）中，并研究其增殖，细胞周期进程，凋亡，菌落形成,和染色体不稳定。使用OVCAR-8和原代HGSOC细胞中的RNA测序分析p53mRNA诱导的转录组改变，其次是独创性途径分析。使用原位异种移植物和转移性腹膜内小鼠模型研究了对肿瘤生长和转移的体内作用。
结果：使用新设计的基于IVTmRNA的方法，在不同的HGSOC模型系统中探索了TP53抑癌基因的再激活。WTp53mRNA的引入引发剂量依赖性细胞凋亡，细胞周期停滞,和有效的持久抑制HGSOC细胞增殖。基于mRNA的p53再激活后OVCAR-8细胞的转录组分析揭示了与p53信号相关的基因表达的显著改变。如细胞凋亡，细胞周期调节，和DNA损伤。同时恢复p53功能可减少HGSOC细胞内的染色体不稳定性，强调其在保护基因组完整性方面的关键贡献，通过缓和由复制应激引起的双链断裂的基线发生。此外，在各种老鼠模型中，p53mRNA治疗以剂量依赖性方式减少了肿瘤生长并抑制了肿瘤细胞在腹膜腔中的传播。
结论：基于IVTmRNA的p53再激活有望成为HGSOC的潜在治疗策略，为p53功能的分子机制及其在卵巢癌治疗中的相关性提供有价值的见解。
