Role of Eya3 in regulating the immune microenvironment to promote breast tumor progression

摘要 Eya蛋白(Eya 1-4)最初被发现是六个家族的基本共激活因子。 同源蛋白通过控制增殖、存活、上皮与胚胎发育 间充质命运，以及在许多组织中的总体细胞命运规范。Six/Eya建筑群是 在发育完成后下调，但在包括乳腺癌在内的许多癌症中重新表达 癌症。除了作为六个蛋白质家族的转录辅助因子外，Eyas还包含一个Tyr 其C-末端结构域中的磷酸酶活性不仅与转录激活有关， 而且还能在运动性、DNA修复和生存方面对损伤、血管生成和抗病毒的抑制作出反应 雌激素受体-b的肿瘤活性。目前，正在开发针对转录和转录因子的抑制剂。 研究Eyas的酪氨酸磷酸酶活性，剖析其不同功能，并开发潜在的抗癌药物 探员们。 EYA在其N-末端结构域中还含有丝氨酸/苏氨酸磷酸酶活性，这是完全可分离的 从酪氨酸磷酸酶活性。这种活性在癌症中的作用是完全未知的，也不是 被任何正在开发的Eya抑制剂靶向。有趣的是，Eya的N-末端结构域不是 含有任何可识别的磷酸酶基序，我们的数据表明，丝氨酸/苏氨酸磷酸酶活性 EYA不是固有的，而是由于与蛋白磷酸酶2A(PP2A)的相互作用。此外, 使用两种不同的免疫能力小鼠模型，我们首次展示了Eya3，即 与其他亚型相比，三阴性乳腺癌(TNBC)中高表达，并包含 最高的丝氨酸/苏氨酸磷酸酶活性，通过其对适应性免疫的影响影响肿瘤的进展。我们 进一步证明，Eya3通过其PP2A相关的丝氨酸/苏氨酸磷酸酶活性，调节关键 免疫检查点蛋白程序性死亡配体1(PD-L1)。因此，我们确定了一种新的肿瘤促进剂 只有在功能性适应性免疫系统的背景下才能揭示Eya3的功能。在本建议书中 我们测试了Eya3通过招募PP2A的能力来调节乳腺肿瘤生长和进展的假设， 导致PD-L1水平升高和肿瘤特异性T细胞反应减弱。 本项目的研究结果将极大地促进我们对Eya3在乳腺肿瘤中的作用的理解 进展，也可能揭示Eya3，通过它以前未知的与PP2A相互作用的能力，是一种 在适应性肿瘤免疫中的重要新参与者。此外，我们可能会揭示一种新的监管机制 关键免疫检查点蛋白PD-L1，其调控在很大程度上仍然难以捉摸。因此，我们的研究可能 导致新的生物标志物或治疗策略，以提高PD-1/PD-L1介导性治疗的疗效。

Abstract The Eya proteins (Eya 1-4) were initially discovered as essential co-activators of the Six family of homeoproteins that regulate embryonic development via controlling proliferation, survival, epithelial versus mesenchymal fates, and overall cell fate specification in numerous tissues. The Six/Eya complex is downregulated after development is complete, but is re-expressed in numerous cancers, including breast cancer. In addition to acting as transcriptional cofactors for the Six family of proteins, Eyas also contain a Tyr phosphatase activity in their C-terminal domains that has been implicated not only in transcriptional activation, but also in motility, DNA repair and survival in response to damage, angiogenesis, and inhibition of the anti- tumor activity of estrogen receptor-b. Currently, inhibitors are being developed against the transcriptional and Tyr phosphatase activities of Eyas to dissect its different functions, and to potentially develop anti-cancer agents. The Eyas also contain a Ser/Thr phosphatase activity in their N-terminal domain, that is completely separable from the Tyr phosphatase activity. The function of this activity in cancer is completely unknown and it is not targeted by any of the Eya inhibitors under development. Intriguingly, the N-terminal domain of Eya does not contain any recognizable phosphatase motifs and our data suggest that the Ser/Thr phosphatase activity of Eya is not intrinsic, but is instead due to an interaction with the protein phosphatase 2A (PP2A). In addition, using two different immune competent mouse models, we show for the first time that Eya3, which is overexpressed in triple negative breast cancers (TNBC) as compared to other subtypes, and contains the highest Ser/Thr phosphatase activity, influences tumor progression via its effects on adaptive immunity. We further demonstrate that Eya3, through its PP2A-associated Ser/Thr phosphatase activity, regulates the key immune checkpoint protein programmed death-ligand 1 (PD-L1). Thus, we identified a novel tumor promoting function for Eya3 that is only revealed in the context of a functional adaptive immune system. In this proposal we test the hypothesis that Eya3 regulates breast tumor growth and progression via its ability to recruit PP2A, leading to increased levels of PD-L1 and a diminished tumor specific T-cell response. Results obtained in this project will significantly advance our understanding of the role of Eya3 in breast tumor progression, and may also reveal that Eya3, through its previously unknown ability to interact with PP2A, is an important new player in adaptive tumor immunity. Further, we may uncover a novel regulatory mechanism of a key immune checkpoint protein, PD-L1, whose regulation has remained largely elusive. Thus our studies may result in novel biomarker or therapeutic strategies to enhance efficacy of PD-1/PD-L1 mediated therapies.