肿瘤抑制因子INPP4B抑制肿瘤细胞衰老的机制及其缺失所诱导的衰老相关分泌性蛋白酶PZP对肿瘤生长的影响

Cellular senescence can occur in cancer cells following exposure to radio- or chemotherapy. This phenomenon is termed as therapy-induced cellular senescence (TIS). TIS is a double-edged sword in cancer treatment because of its senescence associated secretory phenotype (SASP). The mechanisms governing TIS are poorly defined so far. . In a previous functional screening for tumor suppressors in human lung epithelial BEAS-2B cells, we identified inositol polyphosphate 4-phosphatase type II (INPP4B) is a lung tumor suppressor, loss or low expression of INPP4 is often found in lung, breast, ovarian, prostate, as well as many other cancers. To elucidate the mechanism by which INPP4B suppresses the tumorigenesis in lung cells, we generated a cell line with loss of INPP4B protein expression by Crispr-Cas9 gene editing in lung adenocarcinoma A549 cells, and found cellular senescence occurred in a fraction of Crispr-INPP4B cells. Exposure of ionizing radiation (IR, γ-ray ) or DNA damaging drugs to the Crispr-INPP4B cells could enhance such cellular senescence. Moreover, we observed sensitization to DNA damage, and prolonged activation of γ-H2AX and persistent foci formation of Rad51 in the Crispr-INPP4B cells following IR (γ-ray), suggesting homologous recombination (HR) repair is impaired in cells with loss of INPP4B. We also performed quantitative proteomic profiling of the secretome of Crispr-INPP4B senescent cells，and identified protease PZP (pregnancy zone protein) is highly secreted from the INPP4B related senescent cells compared with control cells, indicating that secretion of PZP is the characteristic of INPP4B related senescent cells. . Thus, we propose the impaired DNA damage repair caused by loss of INPP4B is the trigger of the lung cancer cell senescence, and secretion of PZP plays an important role in regulation of tumor microenvironment, tumor growth and metastasis. The aim of our proposal is not only to elucidate the mechanism of cancer cell senescence induced by loss of tumor suppressor INPP4B, but also to provide novel therapeutic targets for selectively killing senescent cancer cells in cancer therapy.

放化疗引起的治疗诱导性肿瘤细胞衰老是影响肿瘤治疗的双刃剑，其调控机制尚不明确。我们前期基因功能筛查发现，磷脂酰肌醇4-磷酸酶Ⅱ型（INPP4B）是一个肺癌相关的肿瘤抑制因子。应用Crispr-Cas9编辑肺腺癌细胞A549中INPP4B基因，造成其蛋白表达完全缺失，导致部分A549细胞发生衰老，电离辐射或化疗药物处理会加重该细胞衰老发生。INPP4B缺失所致衰老与AKT激活无关，而与DNA损伤应答及修复蛋白γ-H2AX、Rad51持续激活相关。定量分泌蛋白组学揭示蛋白酶PZP的大量分泌是该种衰老细胞的分泌表型特征。我们推测DNA同源重组修复能力受损，可能是INPP4B表达缺失细胞发生衰老的诱因，而PZP的大量分泌会影响肿瘤微环境，促进肿瘤生长与侵袭转移。本课题将深入阐明INPP4B缺失所致肿瘤细胞衰老的分子机制和对肿瘤治疗的双刃剑效应，为针对衰老肿瘤细胞的靶向治疗提供理论依据和药物靶点。

我们在国家自然基金资助下，主要进行以下三方面工作.（一） 肿瘤抑制基因INPP4B调控肿瘤细胞衰老的机制.我们发现磷酸肌醇合成通路中的磷脂酰肌醇4-磷酸酶Ⅱ型（INPP4B）是一个潜在的肺癌相关肿瘤抑制基因。我们发现INPP4B蛋白表达缺失A549细胞中自发性衰老比率增高，DNA损伤能促进该类型衰老。为了进一步阐明INPP4B缺失诱导细胞衰老的机制，我们利用免疫共沉淀结合蛋白组学发现肿瘤细胞中INPP4B与DNA双链断裂搭桥蛋白Rad50具有相互作用。INPP4B缺失会导致Rad50的半衰期缩短，γH2AX滞留时间延长，GFP-HR报告系统的同源重组修复效率降低。..（二）定量蛋白质组学刻画电离辐射诱导衰老细胞的全细胞蛋白组学特征.DNA损伤可以诱导细胞衰老，然而, DNA损伤所致衰老细胞的全蛋白组学特征目前仍然不明确。我们采用SILAC（稳定同位素）细胞标记技术，利用定量蛋白组学技术测定DNA损伤诱导的衰老细胞中蛋白丰度变化。我们发现，SASP组分变化是其主要特征，有氧糖酵解下调和ATP合成降低是其特征性表现。我们又发现妊娠区带蛋白PZP在DNA损伤的衰老细胞中高分泌，可以作为一种新型细胞衰老生物标志物。..（三）线粒体靶向化合物SMIP对治疗诱导性衰老肿瘤细胞的选择性杀伤及作用机制.肿瘤细胞经受常规放射治疗或常规化学治疗，部分肿瘤细胞会发生细胞衰老称为治疗诱导性衰老（therapy-induced senescence, TIS）。越来越多研究发现衰老肿瘤细胞是一柄双刃剑。研发选择性清除衰老肿瘤细胞的药物是当前癌症研究的热门领域。我们发现小分子化合物SMIP与已知线粒体靶向能量代谢抑制剂G-boxin在化学结构上相似。通过一系列细胞生物学实验，我们证实：（1）在较高作用浓度时SMIP能够选择性杀伤阿霉素诱导的多种衰老肿瘤细胞，而对生长期肿瘤细胞毒性较低。（2）在较低作用浓度时SMIP能够抑制阿霉素诱导的多种衰老肿瘤细胞的增殖逆转。（3）低浓度SMIP能够抑制ATP产生，促进能量感受器AMPK的激活，增加琥珀酸脱氢酶SDHA、能量应急转录因子ATF4的蛋白表达水平。..本项目执行期间共培养博士研究生2名，硕士研究生5名，其中博士研究生及硕士研究生毕业各1名，已发表本课题相关SCI收录2篇，参加厦门大学主办肿瘤靶向治疗学术会议，发表会议文摘1篇。