Induction of p16Ink4a-dependent senescence through exogenous signals

我们实验室的数据显示，干扰素(干扰素)和肿瘤坏死因子(肿瘤坏死因子)信号通路的同时激活会导致广泛的肿瘤细胞中稳定的细胞周期停滞。这种细胞因子诱导的细胞周期停滞严格依赖于CDK4/6抑制剂p16INK4a，并显示出细胞衰老(CIS)的所有特征。由于肿瘤坏死因子对全身应用来说毒性太大，我们在这里询问是否可以将肿瘤坏死因子信号通路的分子作为靶点。将这些分子与干扰素结合可能会建立一种有效的癌症治疗方法。基于第一个资助期的数据，这项应用有三个相关的项目：(I)寻找当与干扰素结合时诱导癌细胞衰老的分子，我们确定JunB是一个关键的信使，对调节衰老和对干扰素的合成杀伤力很重要。我们已经构建了过表达JunB的肿瘤细胞。令人惊讶的是，单独使用干扰素可以在JunB过度表达的细胞中诱导出CIS。因此，JunB的过表达可以替代CIS所需的肿瘤坏死因子。这不仅为可药物分子开辟了新的途径。更有趣的是，我们的数据强烈表明JunB是一种可调节的分子，它最终决定了干扰素信号是导致生存、衰老还是凋亡。(Ii)寻找可诱导衰老癌细胞凋亡的药物分子(衰老)我们发现，将干扰素与HDAC抑制剂相结合可以诱导衰老。DNA甲基转移酶1(DNMT1)似乎是诱导感官分解的关键分子。现在，我们将通过建立基因敲除细胞(DNMT10/0)，特别是具有结构性DNMT1表达的肿瘤细胞(DNMT1CONST)，来准确地分析DNMT1在顺式反应和感受性降解中的作用。将(DNMT10/0)或(DNMT1CONST)肿瘤细胞(DNMT10/0)或(DNMT1CONST)腹腔注射，然后用带有HDAC抑制剂的干扰素治疗，当与干扰素结合时，将解开DNMT1是否是HDAC抑制剂控制癌症免疫所需的假定的关键信使。(3)一个关键问题涉及衰老癌细胞的自然清除机制，以及这种清除的生物学意义。在这里，我们发现TRAIL和iNOS是能够在体外诱导衰老的天然信号；此外，我们发现衰老的癌细胞需要衰老信号才能被巨噬细胞或树突状细胞清除。现在，重要的是要确定TRAIL和iNOS在体内诱导衰老分解的作用，以及这些分子在体内清除衰老癌症的需要。同时，这些数据将解释免疫系统如何促进癌症的合成致命性，以及当与衰老诱导分子或针对癌症驱动因素的药物结合时，当癌基因诱导的衰老失败时，如何利用这一点来清除癌症。

Data from our laboratory have shown that simultaneous activation of the interferon (IFN) and of the tumor necrosis factor (TNF)-signaling pathways results in a stable cell cycle arrest in a broad range of tumor cells. This cytokine-induced cell cycle arrest strictly depends on the Cdk4/6 inhibitor p16INK4a and shows all characteristic features of cellular senescence (CIS). As TNF is too toxic for systemic applications, here we asked whether molecules of the TNF-signaling pathway could be targeted. Combing such molecules with IFN may allow establishing an efficient cancer therapy. Based on the data gained during the first funding period, this application has three related projects: (I) Searching for molecules that induce senescence in cancer cells when combined with IFN, we identified JunB as a key messenger important for the regulation of senescence and synthetic lethality in response to IFN. We have constructed tumor cells that overexpress JunB. Surprisingly, CIS can be induced in JunB-overexpressing cells with IFN alone. Thus JunB overexpression can replace the TNF required for CIS. This opens not only the approach for a druggable molecule. More interestingly, our data strongly suggest that JunB is a tunable molecule that ultimately determines whether an IFN signal leads to survival, senescence or apoptosis. (II) Searching for druggable molecules that can induce apoptosis in senescent cancer cells (senolysis) we found that combining IFN with HDAC inhibitors can induce senolysis. A key molecule that seems to be critical for the induction of senolysis was DNA-methyl transferase 1 (DNMT1). We will now exactly analyze the role of DNMT1 in CIS and senolysis by establishing knock-out cells (DNMT10/0) and especially also tumor cells with constitutive DNMT1-expression (DNMT1CONST). Intraperitoneal application of (DNMT10/0) or of (DNMT1CONST) tumor cells, followed by treatment with IFN with HDAC inhibitors will unravel whether DNMT1 is the postulated key messenger required for cancer immune control by HDAC inhibitors, when combined with IFN. (III) A key question concerns the natural clearance mechanisms of senescent cancer cells, and the biological meaning of this clearance. Here we identified TRAIL and iNOS as natural signals capable of inducing senolysis in vitro; moreover, we found that senolysis is needed to clear senescent cancer cells by macrophages or dendritic cells. It will now be important to identify the role of TRAIL and iNOS in inducing senolysis in vivo, and the need of these molecules for the in vivo-clearance of senescent cancers.Together, the data will explain how the immune system promotes synthetic lethality in cancers and how this can be exploited to clear cancers, when combined with either senescence-inducing molecules or drugs targeting cancer drivers, when oncogene-induced senescence fails.