IFN-g诱发肺癌细胞衰老改变的分子机制和对肺癌免疫防御系统的影响

IFN-g is the most important cytokine implicated in anti-tumor immunity. Targeting PD-1 and CTLA-4 immune checkpoints have shown remarkable anti-tumor effects. The blockade of PD-1 and CTLA-4 in the treatment of cancers leads to a significant upregulation of IFN-g gene expression. More recent findings suggest that loss of IFN-g associated genes in tumor cells serves as a mechanism of resistance to anti-CTLA-4 and PD-1 blockade therapies in melanoma. It is well known that IFN-g inhibits tumor cell proliferation. Recently, using Agilent microarray, we found that the regulatory molecules of cell cycle including E2F1, Cyclin B1, Cyclin E, which play essential role in promoting cell proliferation, were dramatically downregulated in IFN-g highly expressed tumor tissues compared to the ones expressing low level of IFN-g. Interestingly, growth arrest and DNA damage-24 alpha (GADD45a) was significantly upregulated in IFN-g highly expressed tumor tissues. Besides regulating the cell cycle progression, GADD45A plays a critical role in induction of DNA damage and repair signaling. Our preliminary studies showed that lung cancer cells exposure to IFN-g resulted in increased phosphorylation of H2AX, which is a specific biomarker for DNA damage. Furthermore, IFN-g treated lung cancer cells expressed increased level of phosphorylated ATM. ATM is the key sensor for DNA damage and the key initiator of DNA repair signaling. Our results indicate that IFN-g can induce DNA damage and repair signaling in lung cancer cells. DNA damage and repair signaling not only blocks cell cycle progression but also induces cell senescence. Senescent cells produce multiple types of cytokines including several inflammatory cytokines such as IL-6, IL-8 and IL-1. These inflammatory cytokines can directly inhibit T cell mediated immune response. In addition, IL-6 and IL-8 play critical role in recruiting myeloid derived suppressor cells infiltrating into tumor tissues. As a result, it inhibits adaptive immune responses. In this proposal, we hypothesize that IFN-g induces lung cancer cell DNA damage and repair signaling and results in cell senescence. Cell senescence associated inflammatory cytokines regulate T cell mediated immune function. On the one hand, senescent cells display cell cycle arrest, which represents the anti-tumor effect of IFN-g. On the other hand, cell senescence associated secretory phenotype could affect the T cell function and the status of immune components in tumor microenvironment. Such effect of IFN-g could lead to a new paradigm for tumor immune escape. Investigation of how IFN-g regulates the biological features of tumor cells and how that alternations affect anti-tumor immunity could facilitate identification the key molecule or signaling pathway to manipulate so as to improve the anti-tumor efficacy of IFN-g as well as to prevent the resistance of PD-1 inhibitor based immunotherapy.

IFN-g是抗肿瘤关键效应分子，在肿瘤免疫检查点抑制剂治疗中起到重要作用。IFN-g除了具有广泛的免疫调节作用，还能够直接抑制肿瘤生长。本项目从IFN-g抑制肿瘤增殖这个现象出发，探讨IFN-g抑制肺癌细胞增殖的分子机制以及被IFN-g抑制的这部分肺癌细胞生物学特征和临床意义。前期研究表明肺癌肿瘤组织IFN-g的表达水平不仅和细胞周期通路有关，而且与DNA复制以及p53通路存在联系。最近研究发现IFN-g可以引发肺癌细胞DNA损伤并激活修复通路，还可以诱导肺癌细胞出现衰老样改变。我们推测IFN-g抑制肺癌细胞增殖，同时引发细胞DNA损伤修复，导致部分癌细胞形成衰老细胞。这些衰老细胞产生衰老相关分泌表型等变化，其分泌的炎症因子影响肿瘤免疫微环境和T细胞免疫应答。我们的研究目标是通过系统性研究IFN-g对肿瘤的影响，寻找提高肿瘤免疫治疗疗效和预防治疗耐药发生的关键靶点。

项目背景：细胞因子IFN-γ在肿瘤免疫和肿瘤免疫治疗中起到重要作用，并且是双向作用。IFN-γ抑制肿瘤细胞增殖，但是其分子机制尚不清楚。本项目侄子揭示IFN-γ抑制肿瘤细胞增殖的分子机制及其生物学意义。特别是这些被IFN-γ抑制的肿瘤细胞的归宿和新出现的生物学特征对抗肿瘤免疫应答的影响。.研究内容：.1..揭示IFN-γ抑制肿瘤细胞增殖的机制。.2..揭示IFN-γ诱导肿瘤细胞DNA损伤及其机制。.3..探讨IFN-γ诱导的DNA损伤对肿瘤细胞STING通路的影响。.4..探讨IFN-γ对肿瘤细胞转移能力的影响及其机制。.主要结果：.1..IFN-γ通过引发内质网应激及UPR，抑制肺腺癌细胞增殖，促进细胞凋亡。IFN-γ引发内质网应激依赖STAT1和PI3K-AKT通路。.2..IFN-γ通过上调肿瘤细胞iNOS的表达和一氧化氮的生产，从而诱导细胞发生DNA损伤。.3..IFN-γ激活肿瘤细胞STING通路，上调IFN-β表达。肿瘤细胞胞质dsDNA累积是STING通路激活的关键。IFN-γ诱导的DNA损伤是胞质dsDNA积累的条件。.4..IFN-γ通过激活STAT1/JMJD3上调肿瘤细胞ZEB1表达，从而引发肿瘤细胞EMT，促进肿瘤转移。.科学意义：IFN-γ通过引发内质网应激和UPR调控肿瘤细胞增殖和凋亡。IFN-γ还可以诱导DNA损伤，激活STING通路，从而生产调控肿瘤免疫应答的关键分子IFN-β。由此可见，IFN-γ发挥其抗肿瘤作用可以通过多条途径。这些作用机制之间的关联性还有待进一步挖掘。IFN-γ还有促肿瘤细胞恶性化的作用，表现为引发EMT和促进肿瘤转移。通过对IFN-γ抗癌和促癌作用的进一步认识和机制探讨，有助于寻找和设计联合免疫治疗的新靶点和相应的药物， 为解决当前肿瘤免疫治疗所面临的两个问题，治疗的有效性和持久性，提供理论依据。