线粒体磷酸酶PTPMT1调节小细胞肺癌干细胞代谢及生物学特性研究

The cancer stem cells are located in hypoxic microenvironment and with abnormal energy metabolism. These characteristics are very important for developing novel target therapeutics. Ferroptosis is a recently recognized form of regulated cell death which closely related to energy metabolism and chemo-sensitivity. Our previous works indicated that PTPMT1 is a specific mitochondrial phosphatase closely related to energy metabolism and regulated by hypoxia signaling. However, its expression and roles in regulation of lung cancer stem cells including energy metabolism, ferroptosis and chemo-sensitivity have not been explored. In this study, by analyzing of PTPMT1 in clinical samples of patients with SCLC and PDX models, we will systematically elucidate the roles of PTPMT1 in regulation of lung cancer stem cell characteristics. By using the PTPMT1 silence strategies, we will clarify the roles of PTPMT1 in invasion, proliferation, apoptosis regulation and their mechanisms. We will establish of the assays for ferroptosis and clarify its roles in regulation of ferroptosis and chemo-resistance. The correlation of PTPMT1 expression and SCLC cell chemo-resistance will be explored. In conclusion, this project will elucidate a novel pathophysiological mechanism for SCLS development and provide novel diagnosis and therapeutic targets.

低氧微环境和能量代谢异常是肿瘤干细胞的重要特征，也是肿瘤靶向治疗的重要领域。细胞铁死亡是新近发现的一种细胞死亡方式。我们前期工作发现PTPMT1是线粒体特异并与细胞能量代谢密切相关的磷酸酶，并受低氧信号调控。但其在肺癌干细胞的表达调控及在细胞铁死亡及化疗敏感性的作用和机制并不清楚。本课题通过临床SCLC病人的标本分析和PDX动物模型，系统阐明低氧调控SCLC干细胞调控PTPMT1表达作用的机制；通过基因干涉策略，系统阐明其在SCLC细胞侵袭、增殖、凋亡、恶性转化过程中的作用；建立肺癌细胞铁死亡的检测技术，阐明其对细胞铁死亡的调控作用和机制；阐明PTPMT1和SCLC细胞耐药形成的相关性。本课题将阐明为SCLC病理生理的新机制并为其诊断及治疗提供新的靶点。

本项目主要研究线粒体特异性磷酸酶PTPMT1与小细胞肺癌诊断与治疗的相关性。本项目主要观察了PTPMT1在小细胞肺癌病人标本中表达调控及对肺癌细胞生物学特性的影响。主要进展包括：（1）利用免疫组化检测了SCLC病人标本PTPMT1的表达，发现PTPMT1在SCLC细胞细胞中表达上调。分析SCLC的TCGA数据发现PTPMT1与临床患者的生存率密切相关。PTPMT1高表达SCLC病人的生存期短。因此，我们推论PTPMT1表达与SCLC患者生存率相关。（2）PTPMT1调控SCLC细胞的生物学特性。利用慢病毒介导的shRNA沉默确定了影响肺癌细胞的增殖，PTPMT1沉默后克隆形成减低、凋亡增加并影响细胞迁移等生物学特性。体外实验验证了沉默PTPMT1后抑制A549细胞增殖的结论。（3）建立了SCLC细胞铁死亡的模型，发现Erastin等诱导剂能够诱导SCLC细胞的死亡，其诱导细胞死亡与ROS密切相关，并能够被铁的螯合剂Fer-1逆转，从而确定是理想的铁死亡模型。（4）干涉PTPMT1导致肺癌细胞的糖代谢异常，线粒体功能紊乱，并导致细胞死亡。但PTPMT1抑制导致的细胞死亡并不能够被Fer-1逆转。（5）在PTPMT1处理后细胞RNA测序结果表明，PTPMT1影响肺癌细胞的呼吸链及线粒体内膜蛋白等表达变化；（6）在PTPMT1表达调控中，通过测序发现可变剪切调控分子NUDT21能够调控PTPMT1的可变剪切，NUDT21在SCLC细胞上低表达，并高表达NUDT21能够抑制SCLC细胞的增殖、促进细胞死亡。（7）进一步发现了铁死亡调控基因SENP1，A20，ACSL4和GPX4等在SCLC细胞异常表达，干涉铁死亡调控分子SENP1能够促进肺癌细胞对靶向药物的敏感性。本项目确定了PTPMT1是SCLC治疗的靶点，干涉其调控分子NUDT21和铁死亡调控分子SENP1能够改变肺癌细胞对化疗的敏感性。PTPMT1有望成为肺癌诊断和治疗的靶