Rambow Anna-Christina, Aschenbach Insa, Hagelund Sofie, Tawfik Doaa, Gundlach Jan-Paul, Weiße Sebastian, Maass Nicolai, Trauzold Anna
Department of Gynecology and Obstetrics, University Hospital Schleswig-Holstein (UKSH), Campus Kiel, Kiel, Germany.
Institute for Experimental Cancer Research, University of Kiel, Kiel, Germany.
Front Cell Dev Biol. 2022 Sep 9;10:942718. doi: 10.3389/fcell.2022.942718. eCollection 2022.
Binding of TRAIL to its death domain-containing receptors TRAIL-R1 and TRAIL-R2 can induce cell death and/or pro-inflammatory signaling. The importance of TRAIL and TRAIL-R1/R2 in tumor immune surveillance and cancer biology has meanwhile been well documented. In addition, TRAIL has been shown to preferentially kill tumor cells, raising hope for the development of targeted anti-cancer therapies. Apart from death-inducing receptors, TRAIL also binds to TRAIL-R3 and TRAIL-R4. Whereas TRAIL-R3 is lacking an intracellular domain entirely, TRAIL-R4 contains a truncated death domain but still a signaling-competent intracellular part. It is assumed that these receptors have anti-apoptotic, yet still not well understood regulatory functions. To analyze the significance of the endogenous levels of TRAIL-R4 for TRAIL-induced signaling in cancer cells, we stably knocked down this receptor in Colo357 and MDA-MB-231 cells and analyzed the activation of apoptotic and non-apoptotic pathways in response to treatment with TRAIL. We found that TRAIL-R4 affects a plethora of signaling pathways, partly in an opposite way. While knockdown of TRAIL-R4 in Colo357 strongly increased apoptosis and reduced clonogenic survival, it inhibited cell death and improved clonogenic survival of MDA-MB-231 cells after TRAIL treatment. Furthermore, TRAIL-R4 turned out to be an important regulator of the expression of a variety of anti-apoptotic proteins in MDA-MB-231 cells since TRAIL-R4-KD reduced the cellular levels of FLIPs, XIAP and cIAP2 but upregulated the levels of Bcl-xL. By inhibiting Bcl-xL with Navitoclax, we could finally show that this protein mainly accounts for the acquired resistance of MDA-MB-231 TRAIL-R4-KD cells to TRAIL-induced apoptosis. Analyses of non-apoptotic signaling pathways revealed that in both cell lines TRAIL-R4-KD resulted in a constitutively increased activity of AKT and ERK, while it reduced AKT activity after TRAIL treatment. Furthermore, TRAIL-R4-KD potentiated TRAIL-induced activation of ERK and p38 in Colo357, and NF-κB in MDA-MB-231 cells. Importantly, in both cell lines the activity of AKT, ERK, p38 and NF-κB after TRAIL treatment was higher in TRAIL-R4-KD cells than in respective control cells. Thus, our data provide evidence for the important regulatory functions of endogenous TRAIL-R4 in cancer cells and improve our understanding of the very complex human TRAIL/TRAIL-R system.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）与其含死亡结构域的受体TRAIL-R1和TRAIL-R2结合可诱导细胞死亡和/或促炎信号传导。与此同时，TRAIL和TRAIL-R1/R2在肿瘤免疫监视和癌症生物学中的重要性已得到充分证明。此外，TRAIL已被证明能优先杀死肿瘤细胞，这为开发靶向抗癌疗法带来了希望。除了诱导死亡的受体外，TRAIL还与TRAIL-R3和TRAIL-R4结合。TRAIL-R3完全缺乏细胞内结构域，而TRAIL-R4含有截短的死亡结构域，但仍有一个具有信号传导能力的细胞内部分。据推测，这些受体具有抗凋亡作用，但其调节功能仍未完全了解。为了分析内源性TRAIL-R4水平对癌细胞中TRAIL诱导信号传导的意义，我们在Colo357和MDA-MB-231细胞中稳定敲低该受体，并分析了TRAIL处理后凋亡和非凋亡途径的激活情况。我们发现TRAIL-R4影响众多信号通路，部分作用方式相反。在Colo357细胞中敲低TRAIL-R4可强烈增加细胞凋亡并降低克隆形成存活率，而在TRAIL处理后，它抑制了MDA-MB-231细胞的细胞死亡并提高了克隆形成存活率。此外，TRAIL-R4被证明是MDA-MB-231细胞中多种抗凋亡蛋白表达的重要调节因子，因为TRAIL-R4基因敲除（KD）降低了FLIPs、XIAP和cIAP2的细胞水平，但上调了Bcl-xL的水平。通过用Navitoclax抑制Bcl-xL，我们最终表明该蛋白主要导致MDA-MB-231 TRAIL-R4-KD细胞对TRAIL诱导的凋亡产生获得性抗性。对非凋亡信号通路的分析表明，在这两种细胞系中，TRAIL-R4-KD导致AKT和ERK的活性持续增加，而在TRAIL处理后降低了AKT活性。此外，TRAIL-R4-KD增强了TRAIL诱导的Colo357细胞中ERK和p38的激活，以及MDA-MB-231细胞中NF-κB的激活。重要的是，在这两种细胞系中，TRAIL处理后TRAIL-R4-KD细胞中AKT、ERK、p38和NF-κB的活性高于各自的对照细胞。因此，我们的数据为内源性TRAIL-R4在癌细胞中的重要调节功能提供了证据，并增进了我们对非常复杂的人类TRAIL/TRAIL-R系统的理解。
