Dissecting the activation platform of nuclear caspase-8 in cancer

P53基因突变是肿瘤发展的一个标志，在所有肿瘤中约50%发现，导致这一重要的肿瘤抑制因子功能丧失或获得。因此，癌细胞抵抗依赖于P53的细胞周期检查点和固有的凋亡。有趣的是，剩余50%的肿瘤仍然可以通过野生型p53(Wtp53)进展，这表明这些肿瘤细胞必须能够适应抑制wtp53功能的替代机制。我们最近发现了caspase-8在细胞核中的非典型作用，它允许具有wtp53活性的肿瘤细胞建立事实上的p53蛋白丢失，将细胞命运从细胞周期停滞和凋亡转变为G2/M检查点的有丝分裂细胞分裂。在细胞核中，caspase-8裂解和失活去泛素酶USP28，阻止它去泛素化和稳定wtp53。根据我们最近的发现，抑制核caspase-8可能为这些侵袭性癌症提供了一种理想的替代治疗选择。然而，caspase-8的全面抑制并不是很明显，因为它只会影响含有caspase-8核的肿瘤组织中的一个亚群。相反，它可能对仍表达胞浆caspase-8的肿瘤亚群产生不利影响，旨在诱导细胞死亡。使用三种不同的方法(基于APEX2的邻近生物素化；基于moTAPtag的下拉；全基因组CRISPR-Cas9/sg RNA筛选)，我们的目标是剖析周期和化疗耐药黑色素瘤细胞核中caspase-8激活的平台。这将提高我们对caspase-8/USP28/P53轴在黑色素瘤患者治疗反应中的影响的理解，并可能为确定新的治疗靶点铺平道路。

Mutations in p53 is a hallmark of tumor development and found in ~50% of all tumors, leading to loss-of-function or gain-of-function of this important tumor suppressor. As a consequence, cancer cells resist p53-dependent cell cycle checkpoints and intrinsic apoptosis. Intriguingly, the residual 50% of tumors can still progress with wildtype p53 (wtp53) indicating that these tumor cells must be able to adapt alternative mechanisms suppressing wtp53 functions. We have recently identified a non-canonical role of caspase-8 in the nucleus that allows tumor cells with wtp53 activity to establish a de facto p53 protein loss, shifting cell fate from cell cycle arrest and apoptosis towards mitotic cell division at the G2/M checkpoint. In the nucleus, caspase-8 cleaves and inactivates the deubiquitinase USP28, preventing it from de-ubiquitinating and stabilizing wtp53. Based on our recent finding inhibition of nuclear caspase-8 might provide an ideal alternative treatment option for these aggressive cancers. However, general inhibition of caspase-8 is not indicated because it would only affect a subpopulation within the tumor tissue that harbors nuclear caspase-8. In contrast, it may have adverse effects on tumor subpopulations still expressing cytosolic caspase-8, aiming to induce cell death. Using three different approaches (APEX2-based proximity biotinylation; moTAPtag-based pull down; genome-wide CRISPR-Cas9/sg RNA screen) we aim to dissect the platform of caspase-8 activation in the nucleus of cycling and chemo-resistant melanoma cells. This will improve our understanding about the impact of the caspase-8/USP28/p53 axis in therapy responsiveness of melanoma patients and may pave the way for the identification of new therapeutic targets.